Anti-cd3 antibodies and methods of use

ABSTRACT

The invention provides anti-cluster of differentiation 3 (CD3) antibodies and methods of using the same.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 13, 2021, isnamed 50474-118003_Sequence_Listing_4.13.21_ST25 and is 39,717 bytes insize.

FIELD OF THE INVENTION

The present invention relates to anti-cluster of differentiation 3 (CD3)antibodies and methods of using the same.

BACKGROUND

Cell proliferative disorders, such as cancer, are characterized by theuncontrolled growth of cell subpopulations. They are the leading causeof death in the developed world and the second leading cause of death indeveloping countries, with over 12 million new cancer cases diagnosedand 7 million cancer deaths occurring each year. The American CancerSociety estimates that greater than half a million Americans will die ofcancer in 2015, accounting for nearly one out of every four deaths inthe country. As the elderly population has grown, the incidence ofcancer has concurrently risen, as the probability of developing canceris more than two-fold higher after the age of seventy. Cancer care thusrepresents a significant and ever-increasing societal burden.

Longstanding approaches to cancer treatment include chemotherapy,radiation therapy, and surgery to remove solid tumors. Recently, Tcell-targeting therapeutic antibodies have been developed. Thesetherapeutic antibodies include bispecific antibodies that are capable ofsimultaneously binding cell surface antigens on T cells and cell surfaceantigens on tumor cells, thereby enabling the bound T cells tocontribute to the destruction of the tumor cells.

Existing bispecific antibodies currently undergoing clinical trials fortreating cancer are limited by their short half-lives and/or variableefficacy. Thus, there is an unmet need in the field for the developmentof effective bispecific antibodies for use in cancer treatment.

SUMMARY

The present invention relates to anti-cluster of differentiation 3 (CD3)antibodies and methods of using the same.

In one aspect, the invention features an anti-CD3 antibody, wherein theanti-CD3 antibody comprises a binding domain comprising the followingsix hypervariable regions (HVRs): (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 2; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 3; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 4; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 5; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 6. In some embodiments, the binding domaincomprises (a) a heavy chain variable (VH) domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 7; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 8; or (c) a VH domain as in (a)and a VL domain as in (b). In some embodiments, the VH domain comprisesthe amino acid sequence of SEQ ID NO: 7. In some embodiments, the VLdomain comprises the amino acid sequence of SEQ ID NO: 8. In someembodiments, the binding domain comprises (a) a heavy chain variable(VH) domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 7 and (b) alight chain variable (VL) domain comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 8. In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO: 7. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 8. In some embodiments, theanti-CD3 antibody binds the human CD3ε polypeptide with a K_(D) of 0.7nM or lower (e.g., a K_(D) of 0.6 nM or lower, e.g., a K_(D) of 0.5 nMor lower, e.g., a K_(D) of 0.4 nM or lower, e.g., a K_(D) of 0.3 nM orlower, e.g., a K_(D) of 0.2 nM or lower, e.g., a K_(D) of 0.1 nM orlower).

In some embodiments, any one of the preceding anti-CD3 antibodies can bemonoclonal, human, humanized, or chimeric. In some embodiments, any oneof the preceding anti-CD3 antibodies can be an antibody fragment thatbinds CD3. In some embodiments, the antibody fragment is selected fromthe group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)₂ fragments.In other embodiments, the anti-CD3 antibody is a full-length antibody.In some embodiments, the anti-CD3 antibody is an IgG antibody (e.g., anIgG1, IgG2, or IgG3 antibody). In some embodiments, the anti-CD3antibody is a monospecific antibody. In some embodiments, the anti-CD3antibody is a Bispecific T-Cell Engager (BITE®) antibody).

In some embodiments, the anti-CD3 antibody is a multispecific antibody.In some embodiments, the multispecific antibody is a bispecificantibody, such as a Ly6G6D TDB antibody. In some embodiments, thebispecific antibody comprises a second binding domain that binds to asecond biological molecule, wherein the second biological molecule is acell surface antigen (e.g., a cell surface antigen on a target cellother than an immune effector cell). In some embodiments, the cellsurface antigen is expressed in low copy number on the target cell. Forexample, the cell surface antigen may be expressed at less than 35,000copies per target cell. In some embodiments, the low-copy cell surfaceantigen may be expressed at about 100 copies per target cell to about30,000 copies per target cell.

In some embodiments, the cell surface antigen is a tumor antigen. Insome embodiments, the tumor antigen is selected from the groupconsisting of Ly6G6D (lymphocyte antigen 6 complex, locus G61); Ly6-D,MEGT1); CD20; FcRH5 (Fc Receptor-like 5); HER2; LYPD1; PMEL17 (silverhomolog; SILV; D12S53E; PMEL17; (SI); (SIL); ME20; gp100); Ly6E(lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);CD19; CD33; CD22 (B-cell receptor CD22-B isoform); CD79a (CD79A, CD79a,immunoglobulin-associated alpha; BMPR1 B (bone morphogenetic proteinreceptor-type IB); CD79b (CD79B, CD79β, 1 Gb (immunoglobulin-associatedbeta), B29); EDAR (Ectodysplasin A Receptor); GFRA1 (GDNF-Ra1); MRP4(Multidrug Resistance Protein 4); RET; STEAP1 (six transmembraneepithelial antigen of prostate); TENB2 (putative transmembraneproteoglycan); E16 (LAT1, SLC7A5); 0772P (CA125, MUC16); MPF (MPF, MSLN,SMR, megakaryocyte potentiating factor, mesothelin); Napi2b (NAPI-2B,NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2,type II sodium-dependent phosphate transporter 3b); Sema 5b; PSCA hlg(2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA2700050012 gene); ETBR (Endothelin type B receptor); MSG783 (RNF124,hypothetical protein FLJ20315); STEAP2; TrpM4 (BR22450, FLJ20041, TRPM4,TRPM4B, transient receptor potential cation channel, subfamily M, member4); CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growthfactor); CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barrvirus receptor) or Hs.73792); FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domaincontaining phosphatase anchor protein 1a), SPAP1B, SPAP1C); NCA; MDP;IL20Rα; Brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R (B cell-activatingfactor receptor, BLyS receptor 3, BR3); CXCRS (Burkitt's lymphomareceptor 1; HLA-DOB (Beta subunit of MHC class II molecule); P2X5(Purinergic receptor P2X ligand-gated ion channel 5; CD72 (B-celldifferentiation antigen CD72, Lyb-2); LY64 (Lymphocyte antigen 64(RP105), type I membrane protein of the leucine rich repeat (LRR)family); FcRH1 (Fc receptor-like protein 1); IRTA2 (Immunoglobulinsuperfamily receptor translocation associated 2); TMEFF1; TMEM46 (shisahomolog 2 (Xenopus laevis); SHISA2); LGRS (leucine-richrepeat-containing G protein-coupled receptor 5; GPR49, GPR67); LY6K(lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);GPR19 (G protein-coupled receptor 19; Mm 4787); GPR54 (KISS1 receptor;KISS1R; GPR54; HOT7T175; AXOR12); ASPHD1 (aspartate beta-hydroxylasedomain containing 1; LOC253982); Tyrosinase (TYR; OCAIA; OCA1A;tyrosinase; SHEP3); TMEM118 (ring finger protein, transmembrane 2;RNFT2; FLJ14627); GPR172A (G protein-coupled receptor 172A; GPCR41;FLJ11856; D15Ertd747e); GPC3 (Glypican 3); CLL1 (C-Type Lectin-likemolecule 1); B7-H4 (B7x; B7S1); RNF43 (Ring finger protein 43); CD70;CXORF61 (Chromosome X open reading frame 61); HAVCR1; Epiregulin;Amphiregulin; EGFR; EGFR-L858R; EGFR-L861Q; EGFR-G719A; EGFR-G719S;EGFR-G719C; EGFR-T790M; EGFR-S7681; adipophilin; AIM-2; ALDH1A1;alpha-actinin-4; alpha-foetoprotein; ARTC1; B-RAF; BAGE-1; BCLX (L);BCR-ABL fusion protein (b3a2); beta-catenin; BING-4; CALCA; CASP-5;CASP-8; CD45; Cdc27; CDK4; CDKN2A; CEA; CLPP; COA-1; CPSF; Cw6; cyclinD1; Cyclin-A1; dek-can fusion protein; DKK1; DR1; DR13; EFTUD2;Elongation factor 2; ENAH (hMena); EpCAM; EphA3; ETV6-AML1 fusionprotein; EZH2; FLT3-ITD; FN1; G250; MN; CAIX; GAGE-1;2;8;GAGE-3;4;5;6;7; glypican-3; GnTVf; gp100/Pmel17; GPNMB; HERV-K-MEL;hsp70-2; IDO1; IGF2B3; IL13Ralpha2; Intestinal carboxyl esterase; K-ras;Kallikrein 4; KIF20A; KK-LC-1; KM-HN-1; LAGE-1;LDLR-fucosyltransferaseASfusion protein; Lengsin; M-CSF; MAGE-A1;MAGE-A10; MAGE-A12; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9;MAGE-C1; MAGE-C2; mammaglobin-A; MART2; MCSP; mdm-2; ME1;Melan-A/MART-1; Meloe; MMP-2; MMP-7; MUC1; MUC5AC; mucin; MUM-1f; MUM-2;MUM-3; Myosin class I; N-ras; NA88-A; neo-PAP; NFYC; NY-BR-1;NY-ESO-1/LAGE-2; OA1; OGT; OS-9; p53; PAP; PAXS; PBF; pml-RARalphafusion protein; PRAME; PRDXS; PSMA; PTPRK; RAB38/NY-MEL-1; RAGE-1;RBAF600; RGSS; RhoC; RNF43; RU2AS; SAGE; secernin 1; SIRT2; SNRPD1;SOX10; Sp17; SSX-2; SSX-4; STEAP1; survivin; SYT-SSX1 or -SSX2 fusionprotein; TAG-1; TAG-2; Telomerase; TGF-betaRII; TRAG-3; Triosephosphateisomerase; TRP-1/gp75; TRP-2; TRP2-INT2; tyrosinase; VEGF; WT1; XAGE-1b/GAGED2a; and SLC35D3. In some embodiments, the tumor antigen isselected from the group consisting of LY6G6D, CD20, FcRH5, HER2, LYPD1,PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET,Steap1, and TenB2.

In some embodiments, the tumor antigen is Ly6G6D, which may be presenton the cell surface of the target cell at between about 20,000 copiesper target cell to about 30,000 copies per target cell. In someembodiments, copy number may be determined by Scatchard plot analysis.

In a related aspect, the invention features an anti-CD3 antibody,wherein the anti-CD3 antibody is a bispecific antibody that binds to CD3located on an immune effector cell and a cell surface antigen that isexpressed in low copy number on a target cell other than the immuneeffector cell, wherein the bispecific antibody comprises an anti-CD3 armcomprising a first binding domain comprising the following six HVRs: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 5; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 6; and an anti-cellsurface antigen arm comprising a second binding domain. In someembodiments, the cell surface antigen is a tumor antigen. In someembodiments, the tumor antigen is Ly6G6D.

In some embodiments, any one of the preceding anti-CD3 antibodies maycomprise a substitution mutation in the Fc region that reduces effectorfunction. In some embodiments, the substitution mutation is anaglycosylation site mutation. In some embodiments, the aglycosylationsite mutation is at amino acid residue N297, L234, L235, and/or D265 (EUnumbering). In some embodiments, the aglycosylation site mutation isselected from the group consisting of N297G, N297A, L234A, L235A, andD265A. In some embodiments, the mutation is an N297G mutation. In someembodiments, the aglycosylation site mutation reduces effector functionof the anti-CD3 antibody.

In some embodiments, any one of the preceding anti-CD3 antibodies cancomprise one or more heavy chain constant domains, wherein the one ormore heavy chain constant domains are selected from a first CH1 (CH1₁)domain, a first CH2 (CH2₁) domain, a first CH3 (CH3₁) domain, a secondCH1 (CH1₂) domain, second CH2 (CH2₂) domain, and a second CH3 (CH3₂)domain. In some embodiments, at least one of the one or more heavy chainconstant domains is paired with another heavy chain constant domain. Insome embodiments, the CH3₁ and CH3₂ domains each comprise a protuberanceor cavity, and wherein the protuberance or cavity in the CH3, domain ispositionable in the cavity or protuberance, respectively, in the CH3₂domain. In some embodiments, the CH3₁ and CH3₂ domains meet at aninterface between said protuberance and cavity. In some embodiments, theCH2₁ and CH2₂ domains each comprise a protuberance or cavity, andwherein the protuberance or cavity in the CH2, domain is positionable inthe cavity or protuberance, respectively, in the CH2₂ domain. In someembodiments, the CH2₁ and CH2₂ domains meet at an interface between saidprotuberance and cavity.

In some embodiments, the invention features an immunoconjugatecomprising any one of the preceding anti-CD3 antibodies conjugated to acytotoxic agent. Also provided is a composition comprising any one ofthe preceding anti-CD3 antibodies. In some embodiments, the compositionfurther comprises a pharmaceutically acceptable carrier, excipient, ordiluent. In some embodiments, the composition is a pharmaceuticalcomposition. In some embodiments, the composition further comprises aPD-1 axis binding antagonist or an additional therapeutic agent. Inanother aspect, the invention features an isolated nucleic acid thatencodes any of the anti-CD3 antibodies disclosed herein. In someembodiments, a vector (e.g., an expression vector) comprising thenucleic acid encoding an anti-CD3 antibody is provided (e.g., a vectorfor expressing the anti-CD3 antibody).

In another aspect, the invention features host cells comprising thepreceding nucleic acids and/or vectors. In some embodiments, the hostcell is a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell). Inother embodiments, the host cell is a prokaryotic cell (e.g., an E. colicell). A method of producing any one of the preceding anti-CD3antibodies is also provided, the method comprising culturing the hostcell that produces the anti-CD3 antibody and recovering the anti-CD3antibody from the host cell or the culture medium.

In some aspects, any one of the preceding anti-CD3 antibodies can be foruse as a medicament. In some embodiments, any one of the precedinganti-CD3 antibodies can be for use in treating or delaying progressionof a cell proliferative disorder or an autoimmune disorder in a subjectin need thereof. In some embodiments, any one of the preceding anti-CD3antibodies can be for use in enhancing immune function in a subjecthaving a cell proliferative disorder or an autoimmune disorder.

In some aspects, the invention features the use of any one of thepreceding anti-CD3 antibodies in the manufacture of a medicament fortreating or delaying progression of a cell proliferative disorder or anautoimmune disorder. In some aspects, the invention features the use ofany one of the preceding anti-CD3 antibodies in the manufacture of amedicament for enhancing immune function in a subject having a cellproliferative disorder or an autoimmune disorder.

A further aspect of the invention is a method of treating or delayingthe progression of a cell proliferative disorder or an autoimmunedisorder in a subject in need thereof, the method comprisingadministering to the subject an effective amount any one of thepreceding anti-CD3 antibodies. In another aspect, the invention featuresa method of enhancing immune function in a subject having a cellproliferative disorder or an autoimmune disorder, the method comprisingadministering to the subject any one of the preceding anti-CD3antibodies. In some embodiments, the anti-CD3 antibody binds to (a) aCD3 molecule located on an immune effector cell and (b) a secondbiological molecule located on a target cell other than the immuneeffector cell. In some embodiments, the anti-CD3 antibody activates theimmune effector cell following binding to (a) and (b). In someembodiments, the activated immune effector cell is capable of exerting acytotoxic effect and/or an apoptotic effect on the target cell. In someembodiments, the anti-CD3 antibody is administered to the subject in adosage of about 0.01 mg/kg to about 10 mg/kg. In some embodiments, theanti-CD3 antibody is administered to the subject in a dosage of about0.1 mg/kg to about 10 mg/kg. In some embodiments, the anti-CD3 antibodyis administered to the subject in a dosage of about 1 mg/kg. In someembodiments, the anti-CD3 antibody is administered subcutaneously,intravenously, intramuscularly, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the anti-CD3 antibody is administered subcutaneously. In someembodiments, the anti-CD3 antibody is administered intravenously.

In some embodiments, the method further comprises administering to thesubject a PD-1 axis binding antagonist or an additional therapeuticagent. In some embodiments, the additional therapeutic agent isadministered prior to or subsequent to the administration of theanti-CD3 antibody. In some embodiments, the additional therapeutic agentis administered concurrently with the anti-CD3 antibody. In someembodiments, the PD-1 axis binding antagonist is selected from the groupconsisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, anda PD-L2 binding antagonist.

In some embodiments, the PD-1 axis binding antagonist is a PD-1 bindingantagonist. In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody, antigen-binding fragment thereof, immunoadhesin,fusion protein, oligopeptide, or other molecule that decreases, blocks,inhibits, abrogates, or interferes with signal transduction resultingfrom the interaction of PD-1 with PD-L1 and/or PD-L2. In someembodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In aspecific embodiment, the PD-1 binding antagonist is MDX-1106(nivolumab). In another specific embodiment, the PD-1 binding antagonistis MK-3475 (pembrolizumab). In another specific embodiment, the PD-1binding antagonist is CT-011 (pidilizumab). In another specificembodiment, the PD-1 binding antagonist is AMP-224. In another specificembodiment, the PD-1 binding antagonist is MED1-0680. In anotherspecific embodiment, the PD-1 binding antagonist is PDR001. In anotherspecific embodiment, the PD-1 binding antagonist is REGN2810. In anotherspecific embodiment, the PD-1 binding antagonist is BGB-108.

In other embodiments, the PD-1 axis binding antagonist is a PD-L1binding antagonist. In some embodiments, the PD-L1 binding antagonist isan anti-PD-L1 antibody, antigen binding fragment thereof, immunoadhesin,fusion protein, oligopeptide, or other molecule that decreases, blocks,inhibits, abrogates, or interferes with signal transduction resultingfrom the interaction of PD-L1 with one or more of its binding partners,such as PD-1, B7-1. In some embodiments, the PD-L1 binding antagonist isan anti-PD-L1 antibody. In still another specific embodiment, theanti-PD-L1 antibody is MPDL3280A (atezolizumab). In a specificembodiment, the anti-PD-L1 antibody is YW243.55.S70. In another specificembodiment, the anti-PD-L1 antibody is MDX-1105. In another specificembodiment, the anti PD-L1 antibody is MSB0015718C. In still anotherspecific embodiment, the anti-PD-L1 antibody is MEDI4736.

In other embodiments, the PD-1 axis binding antagonist is a PD-L2binding antagonist. In some embodiments, the PD-L2 binding antagonist isan anti-PD-L2 antibody, antigen binding fragment thereof, immunoadhesin,fusion protein, oligopeptide, or other molecule that decreases, blocks,inhibits, abrogates, or interferes with signal transduction resultingfrom the interaction of PD-L2 with one or more of its binding partners,such as PD-1. In some embodiments, the PD-L2 binding antagonist is ananti-PD-L2 antibody. In some embodiments, the PD-L2 binding antagonistis an immunoadhesin.

In some aspects, the invention features a method of treating or delayingthe progression of a cell proliferative disorder or an autoimmunedisorder in a subject in need thereof, the method comprisingadministering to the subject an anti-CD3 antibody and a PD-1 axisbinding antagonist, wherein the anti-CD3 antibody comprises an anti-CD3arm and an anti-Ly6G6D arm. In some aspects, the invention features amethod of enhancing immune function in a subject having a cellproliferative disorder or an autoimmune disorder, the method comprisingadministering to the subject an anti-CD3 antibody and a PD-1 axisbinding antagonist, wherein the anti-CD3 antibody comprises an anti-CD3arm and an anti-Ly6G6D arm (i.e., a Ly6G6D TDB antibody). In someembodiments, the anti-CD3 arm comprises a first binding domaincomprising (i) a VH domain comprising an amino acid sequence of SEQ IDNO: 7, and (ii) a VL domain comprising an amino acid sequence of SEQ IDNO: 8. In some embodiments, the method includes administering to thesubject an anti-CD3 antibody, wherein the anti-CD3 antibody comprises(i) an anti-CD3 arm having a VH domain comprising an amino acid sequenceof SEQ ID NO: 7 and a VL domain comprising an amino acid sequence of SEQID NO: 8 and (ii) an anti-Ly6G6D arm (i.e., a Ly6G6D TDB antibody), anda PD-1 axis binding antagonist that is an anti-PD-L1 antibody.

In other embodiments, an anti-CD3 antibody (e.g., a Ly6G6D TDB) isco-administered (concurrently, as a single or multiple (e.g., 1, 2, 3,4, 5, or 6 or more) compositions (e.g., formulations)) with one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) additional therapeuticagents selected from FOLFOX (oxaliplatin (ELOXATIN™) combined with5-fluorouracil and leucovorin), capecitabine (XELODA®), 5-fluorouracil(5-FU), CapeOx (XELOX; capecitabine with oxaliplatin), leucovorin(folinic acid), bevacizumab (AVASTIN®), cetuximab (ERBITUX®),panitumumab (VECTIBIX®), regorafenib (STIVARGA®), irinotecan (CPT-11;CAMPTOSAR®), and FLOX (5-fluorouracil with oxaliplatin). In otherembodiments, an anti-CD3 antibody (e.g., a Ly6G6D TDB) is administeredbefore one or more additional therapeutic agents, such as any one, two,three, four, five, six, seven, eight, nine, ten, or all eleven of thefollowing: FOLFOX (oxaliplatin (ELOXATIN™) combined with 5-fluorouraciland leucovorin), capecitabine (XELODA®), 5-fluorouracil (5-FU), CapeOx(XELOX; capecitabine with oxaliplatin), leucovorin (folinic acid),bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®),regorafenib (STIVARGA®), irinotecan (CPT-11; CAMPTOSAR®), and FLOX(5-fluorouracil with oxaliplatin). In other embodiments, an anti-CD3antibody (e.g., a Ly6G6D TDB) is administered after one or moreadditional therapeutic agents, such as any one, two, three, four, five,six, seven, eight, nine, ten, or all eleven of the following: FOLFOX(oxaliplatin (ELOXATIN™) combined with 5-fluorouracil and leucovorin),capecitabine (XELODA®), 5-fluorouracil (5-FU), CapeOx (XELOX;capecitabine with oxaliplatin), leucovorin (folinic acid), bevacizumab(AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), regorafenib(STIVARGA®), irinotecan (CPT-11; CAMPTOSAR®), and FLOX (5-fluorouracilwith oxaliplatin).

In some embodiments, the method further comprises administering to thesubject a glucocorticoid. In some embodiments, the glucocorticoid isselected from the group consisting of dexamethasone, hydrocortisone,cortisone, prednisolone, prednisone, methylprednisone, triamcinolone,paramethasone, betamethasone, fludrocortisone, and pharmaceuticallyacceptable esters, salts, and complexes thereof. In some embodiments,the glucocorticoid is dexamethasone. In some embodiments, theglucocorticoid is a pharmaceutically acceptable ester, salt, or complexof dexamethasone.

In some embodiments, the method further comprises administering to thesubject rituximab. In some embodiments, the method further comprisesadministering to the subject obinutuzumab. In some embodiments, themethod further comprises administering to the subject an antibody-drugconjugate (ADC).

In any of the preceding uses or methods, the cell proliferative disordercan be cancer. In some embodiments, the cancer is selected from thegroup consisting of esophageal cancer, stomach cancer, small intestinecancer, large intestine cancer, colorectal cancer, breast cancer,non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B celllymphoma, B cell leukemia, multiple myeloma, renal cancer, prostatecancer, liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, glioblastoma, germinal-center B-cell-like (GCB) DLBCL,activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle celllymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia(CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL),lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM),central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cellprolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cellleukemia, Splenic lymphoma/leukemia, unclassifiable, Splenic diffuse redpulp small B-cell lymphoma, Hairy cell leukemia variant, WaldenstrOmmacroglobulinemia, Heavy chain diseases, a Heavy chain disease, γ Heavychain disease, μ Heavy chain disease, Plasma cell myeloma, Solitaryplasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginalzone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma),Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma,Pediatric follicular lymphoma, Primary cutaneous follicle centrelymphoma, T-cell/histiocyte rich large B-cell lymphoma, Primary DLBCL ofthe CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of theelderly, DLBCL associated with chronic inflammation, Lymphomatoidgranulomatosis, Primary mediastinal (thymic) large B-cell lymphoma,Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma,Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, Primary effusion lymphoma: B-celllymphoma, unclassifiable, with features intermediate between diffuselarge B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma,unclassifiable, with features intermediate between diffuse large B-celllymphoma and classical Hodgkin lymphoma.

In some embodiments, the cancer is esophageal cancer. In someembodiments, the cancer is an adenocarcinoma, for example, a metatstaticadenocarcinoma (e.g., a colorectal adenocarcinoma, a gastricadenocarcinoma, or a pancreatic adenocarcinoma).

In any of the preceding uses or methods, the autoimmune disorder can beselected from the group consisting of rheumatoid arthritis, juvenilerheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener'sdisease, inflammatory bowel disease, idiopathic thrombocytopenic purpura(ITP), thrombotic thrombocytopenic purpura (TTP), autoimmunethrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgMpolyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,Reynaud's syndrome, Sjorgen's syndrome, glomerulonephritis,Neuromyelitis Optica (NMO), and IgG neuropathy.

In another aspect, the invention features a kit comprising: (a) acomposition comprising any one of the preceding anti-CD3 antibodies and(b) a package insert comprising instructions for administering thecomposition to a subject to treat or delay progression of a cellproliferative disorder.

In any of the preceding uses or methods, the subject can be a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the amino acid sequence of the light chain variable domain(VL) of the anti-CD3 antibody 38E4v11 (SEQ ID NO: 8). HVR-L1 (SEQ ID NO:4), HVR-L2 (SEQ ID NO: 5), and HVR-L3 (SEQ ID NO: 6) sequences aredelimited by the denoted boxes.

FIG. 1B shows the amino acid sequence of the heavy chain variable domain(VH) of the anti-CD3 antibody 38E4v11 (SEQ ID NO: 7). HVR-H1 (SEQ ID NO:1), HVR-H2 (SEQ ID NO: 2), and HVR-H3 (SEQ ID NO: 3) sequences aredelimited by the denoted boxes.

FIG. 2 is a graph showing the percentage of Ly6G6D-transfected 293target cells killed in an in vitro target cell killing assay as afunction of Ly6G6D TDB antibody concentration for three different Ly6G6DTDBs having different anti-CD3 arms: Ly6G6D (38E4v11) TDB, Ly6G6D(40G5c) TDB, and Ly6G6D (38E4v1) TDB.

FIG. 3A is a graph showing the percentage of CD69+CD25+ activated CD8+ Tcells as a function of Ly6G6D TDB concentration for three differentLy6G6D TDBs (Ly6G6D (38E4v11) TDB, Ly6G6D (40G5c) TDB, and Ly6G6D(38E4v1) TDB), as assessed by flow cytometry analysis. Effector cell:target cell ratio=5:1.

FIG. 3B is a graph showing the percentage of CD69+CD25+ activated CD4+ Tcells as a function of Ly6G6D TDB concentration for three differentLy6G6D TDBs (Ly6G6D (38E4v11) TDB, Ly6G6D (40G5c) TDB, and Ly6G6D(38E4v1) TDB), as assessed by flow cytometry analysis. Effector cell:target cell ratio=5:1.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-CD3 antibody” and “an antibody that binds to CD3” referto an antibody that is capable of binding CD3 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD3. In one embodiment, the extent of binding of ananti-CD3 antibody to an unrelated, non-CD3 protein is less than about10% of the binding of the antibody to CD3 as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody that bindsto CD3 has a dissociation constant (K_(D)) of 1 μM, 100 nM, 10 nM, 1 nM,0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸M to10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments, ananti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments.

By “binding domain” is meant a part of a compound or a molecule thatspecifically binds to a target epitope, antigen, ligand, or receptor.Binding domains include but are not limited to antibodies (e.g.,monoclonal, polyclonal, recombinant, humanized, and chimericantibodies), antibody fragments or portions thereof (e.g., Fabfragments, Fab′₂, scFv antibodies, SMIP, domain antibodies, diabodies,minibodies, scFv-Fc, affibodies, nanobodies, and VH and/or VL domains ofantibodies), receptors, ligands, aptamers, and other molecules having anidentified binding partner.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); combretastatin; folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®,Bristol-Myers Squibb Oncology, Princeton, N.J.), albumin-engineerednanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel(TAXOTERE®, Rhome-Poulene Rorer, Antony, France); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum agents such ascisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, whichprevent tubulin polymerization from forming microtubules, includingvinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®,FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide;mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin;aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid,including bexarotene (TARGRETIN®); bisphosphonates such as clodronate(for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095,zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R) (e.g., erlotinib (Tarceva™)); and VEGF-A that reduce cellproliferation; vaccines such as THERATOPE® vaccine and gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH(e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib,SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib oretoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®);CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosinekinase inhibitors; serine-threonine kinase inhibitors such as rapamycin(sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such aslonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON.cndot.toremifene; aromataseinhibitors that inhibit the enzyme aromatase, which regulates estrogenproduction in the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, andARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,particularly those which inhibit expression of genes in signalingpathways implicated in abherant cell proliferation, such as, forexample, PKC-alpha, Raf and H-Ras; ribozymes such as a VEGF expressioninhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor;vaccines such as gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2;LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine andEsperamicins (see U.S. Pat. No. 4,675,187), and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The term “cluster of differentiation 3” or “CD3,” as used herein, refersto any native CD3 from any vertebrate source, including mammals such asprimates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated, including, for example, CD3ε, CD3γ, CD3α, and CD3βchains. The term encompasses “full-length,” unprocessed CD3 (e.g.,unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of CD3, including, for example, splice variants orallelic variants. CD3 includes, for example, human CD3ε protein (NCBIRefSeq No. NP 000724), which is 207 amino acids in length, and humanCD3γ protein (NCBI RefSeq No. NP 000064), which is 182 amino acids inlength.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

A “disorder” is any condition that would benefit from treatmentincluding, but not limited to, chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer. In one embodiment, the cell proliferative disorder is a tumor.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, adenocarcinoma (e.g., colorectal adenocarcinoma, gastricadenocarcinoma, or pancreatic adenocarcinoma), which may be metastaticadenocarcinoma (e.g., metastatic colorectal adenocarcinoma, metastaticgastric adenocarcinoma, or metastatic pancreatic adenocarcinoma),carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include, but arenot limited to, esophageal cancer, small intestine cancer, largeintestine cancer, squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer and gastrointestinalstromal cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, cancer of the urinarytract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma,superficial spreading melanoma, lentigo maligna melanoma, acrallentiginous melanomas, nodular melanomas, multiple myeloma and B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), Meigs' syndrome,brain, as well as head and neck cancer, and associated metastases. Incertain embodiments, cancers that are amenable to treatment by theantibodies of the invention include adenocarcinomas (e.g., colorectaladenocarcinoma, gastric adenocarcinoma, or pancreatic adenocarcinoma),which may be metastatic adenocarcinomas (e.g., metastatic colorectaladenocarcinoma, metastatic gastric adenocarcinoma, or metastaticpancreatic adenocarcinoma), esophageal cancer, stomach cancer, smallintestine cancer, large intestine cancer, colorectal cancer, breastcancer, colorectal cancer, rectal cancer, non-small cell lung cancer,glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostatecancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi'ssarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer,mesothelioma, and multiple myeloma. In some embodiments, the cancer isselected from: adenocarcinomas (e.g., colorectal adenocarcinoma, gastricadenocarcinoma, or pancreatic adenocarcinoma), which may be metastaticadenocarcinomas (e.g., metastatic colorectal adenocarcinoma, metastaticgastric adenocarcinoma, or metastatic pancreatic adenocarcinoma),esophageal cancer, stomach cancer, small intestine cancer, largeintestine cancer, small cell lung cancer, gliblastoma, neuroblastomas,melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), andhepatocellular carcinoma. Yet, in some embodiments, the cancer isselected from: adenocarcinomas (e.g., colorectal adenocarcinoma, gastricadenocarcinoma, or pancreatic adenocarcinoma), esophageal cancer,stomach cancer, small intestine cancer, large intestine cancer,non-small cell lung cancer, colorectal cancer, glioblastoma and breastcarcinoma, including metastatic forms of those cancers. In otherembodiments, the cancer is selected from a class of mature B-Cellcancers excluding Hodgkin's Lymphoma but including germinal-centerB-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicularlymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML),chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), smalllymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrommacroglobulinemia (WM), central nervous system lymphoma (CNSL),Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, Splenicmarginal zone lymphoma, Hairy cell leukemia, Splenic lymphoma/leukemia,unclassifiable, Splenic diffuse red pulp small B-cell lymphoma, Hairycell leukemia variant, WaldenstrOm macroglobulinemia, Heavy chaindiseases, a Heavy chain disease, γ Heavy chain disease, μ Heavy chaindisease, Plasma cell myeloma, Solitary plasmacytoma of bone,Extraosseous plasmacytoma, Extranodal marginal zone lymphoma ofmucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zonelymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicularlymphoma, Primary cutaneous follicle centre lymphoma, T-cell/histiocyterich large B-cell lymphoma, Primary DLBCL of the CNS, Primary cutaneousDLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associatedwith chronic inflammation, Lymphomatoid granulomatosis, Primarymediastinal (thymic) large B-cell lymphoma, Intravascular large B-celllymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma,Large B-cell lymphoma arising in HHV8-associated multicentric Castlemandisease, Primary effusion lymphoma: B-cell lymphoma, unclassifiable,with features intermediate between diffuse large B-cell lymphoma andBurkitt lymphoma, and B-cell lymphoma, unclassifiable, with featuresintermediate between diffuse large B-cell lymphoma and classical Hodgkinlymphoma.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder,” and “tumor” are notmutually exclusive as referred to herein.

The term “tumor antigen,” as used herein, may be understood as thoseantigens that are presented on tumor cells. These antigens can bepresented on the cell surface with an extracellular part, which is oftencombined with a transmembrane and cytoplasmic part of the molecule.These antigens can sometimes be presented only by tumor cells and neverby the normal ones. Tumor antigens can be exclusively expressed on tumorcells or might represent a tumor-specific mutation compared to normalcells. In this case, they are caned tumor-specific antigens. More commonare tumor antigens that are presented by tumor cells and normal cells,and they are caned tumor-associated antigens. These tumor-associatedantigens can be overexpressed compared to normal cells or are accessiblefor antibody binding in tumor cells due to the less compact structure ofthe tumor tissue compared to normal tissue. Tumor antigens may exhibitinconsistent expression or may be expressed in low copy number oncertain types of tumor cells. In instances in which a targeted tumorantigen is expressed in low copy number (i.e., weakly expressed) ontumor cells, it may be desirable to use a TDB antibody of the inventionhaving a high-affinity arm against CD3 or another molecule located on animmune effector cell. In one aspect, the tumor antigen is selected fromthose set forth in Table 1 below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1 q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of a compound, for example, an anti-CD3 antibodyof the invention or a composition (e.g., pharmaceutical composition)thereof, is at least the minimum amount required to achieve the desiredtherapeutic or prophylactic result, such as a measurable improvement orprevention of a particular disorder (e.g., a cell proliferativedisorder, e.g., cancer). An effective amount herein may vary accordingto factors such as the disease state, age, sex, and weight of thepatient, and the ability of the antibody to elicit a desired response inthe individual. An effective amount is also one in which any toxic ordetrimental effects of the treatment are outweighed by thetherapeutically beneficial effects. For prophylactic use, beneficial ordesired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. In the case ofcancer or tumor, an effective amount of the drug may have the effect inreducing the number of cancer cells; reducing the tumor size; inhibiting(i.e., slow to some extent or desirably stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent and desirablystop) tumor metastasis; inhibiting to some extent tumor growth; and/orrelieving to some extent one or more of the symptoms associated with thedisorder. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofdrug, compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. As is understood in the clinical context, an effectiveamount of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective amount” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.In one embodiment, growth inhibitory agent is growth inhibitory antibodythat prevents or reduces proliferation of a cell expressing an antigento which the antibody binds. In another embodiment, the growthinhibitory agent may be one which significantly reduces the percentageof cells in S phase. Examples of growth inhibitory agents include agentsthat block cell cycle progression (at a place other than S phase), suchas agents that induce G1 arrest and M-phase arrest. Classical M-phaseblockers include the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in Mendelsohn and Israel, eds., The Molecular Basis of Cancer,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W.B. Saunders, Philadelphia,1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) areanticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®,Rhone-Poulenc Rorer), derived from the European yew, is a semisyntheticanalogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel anddocetaxel promote the assembly of microtubules from tubulin dimers andstabilize microtubules by preventing depolymerization, which results inthe inhibition of mitosis in cells.

The term “HER2-positive” cancer comprises cancer cells which have higherthan normal levels of HER2. Examples of HER2-positive cancer includeHER2-positive breast cancer and HER2-positive gastric cancer.Optionally, HER2-positive cancer has an immunohistochemistry (IHC) scoreof 2+ or 3+ and/or an in situ hybridization (ISH) amplification ratio2.0.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. Human antibodies can beproduced using various techniques known in the art, includingphage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available forthe preparation of human monoclonal antibodies are methods described inCole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See alsovan Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001).Human antibodies can be prepared by administering the antigen to atransgenic animal that has been modified to produce such antibodies inresponse to antigenic challenge, but whose endogenous loci have beendisabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181and 6,150,584 regarding XENOMOUSE™ technology). See also, for example,Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regardinghuman antibodies generated via a human B-cell hybridoma technology.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

A “subject” or an “individual” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, thesubject or individual is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-CD3 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “Ly6G6D” or “lymphocyte antigen 6 complex, locus G61,” as usedherein, refers to any native Ly6G6D from any vertebrate source,including mammals such as primates (e.g. humans) and rodents (e.g., miceand rats), unless otherwise indicated, and encompasses “full-length,”unprocessed Ly6G6D, as well as any form of Ly6G6D that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of Ly6G6D, including, for example, splice variants or allelicvariants. Ly6G6D is also referred to as G6D, Ly6-D, C6orf23,megakaryocyte-enhanced gene transcript 1 (MEGT1), and NG25 and isdisclosed in U.S. Pat. No. 7,951,546, which is incorporated by referenceherein in its entirety, as TAT201, with an amino acid sequence of SEQ IDNO: 92 and a nucleotide sequence, DNA234441, of SEQ ID NO: 36. Ly6G6Dincludes, for example, human Ly6G6D protein (NCBI RefSeq No.NP_067079.2), which is 133 amino acids in length.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (A), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partners, so as to remove T cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific embodiment, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes, such assignaling mediated through PD-1, so as render a dysfunctional T cellless dysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific embodiment, a PD-1 binding antagonistis MDX-1106 (nivolumab). In another specific embodiment, a PD-1 bindingantagonist is MK-3475 (pembrolizumab). In another specific embodiment, aPD-1 binding antagonist is CT-011 (pidilizumab). In another specificembodiment, a PD-1 binding antagonist is AMP-224. In another specificembodiment, a PD-1 binding antagonist is MED1-0680. In another specificembodiment, a PD-1 binding antagonist is PDR001. In another specificembodiment, a PD-1 binding antagonist is REGN2810. In another specificembodiment, a PD-1 binding antagonist is BGB-108.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific embodiment, the PD-L1 bindingantagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In someembodiments, the PD-L1 binding antagonists include anti-PD-L1antibodies, antigen binding fragments thereof, immunoadhesins, fusionproteins, oligopeptides and other molecules that decrease, block,inhibit, abrogate or interfere with signal transduction resulting fromthe interaction of PD-L1 with one or more of its binding partners, suchas PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces thenegative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes, such as signaling mediated throughPD-L1, so as to render a dysfunctional T cell less dysfunctional (e.g.,enhancing effector responses to antigen recognition). In someembodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. Instill another specific embodiment, an anti-PD-L1 antibody is MPDL3280A(atezolizumab). In a specific embodiment, an anti-PD-L1 antibody isYW243.55.S70. In another specific embodiment, an anti-PD-L1 antibody isMDX-1105. In another specific embodiment, an anti PD-L1 antibody isMSB0015718C. In still another specific embodiment, an anti-PD-L1antibody is MEDI4736.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific embodiment, the PD-L2binding antagonist inhibits binding of PD-L2 to PD-1. In someembodiments, the PD-L2 antagonists include anti-PD-L2 antibodies,antigen binding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-L2 with either one or more of its binding partners,such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces thenegative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes, such as signaling mediated throughPD-L2, so as render a dysfunctional T cell less dysfunctional (e.g.,enhancing effector responses to antigen recognition). In someembodiments, a PD-L2 binding antagonist is an immunoadhesin.

The term “protein,” as used herein, refers to any native protein fromany vertebrate source, including mammals such as primates (e.g. humans)and rodents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed protein as well as any form ofthe protein that results from processing in the cell. The term alsoencompasses naturally occurring variants of the protein, e.g., splicevariants or allelic variants. Proteins according to the inventioninclude, for example, any protein listed in Table 1.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

As used herein, “delaying progression” of a disorder or disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease or disorder (e.g., a cell proliferative disorder, e.g.,cancer). This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. For example, a late stage cancer, such as development ofmetastasis, may be delayed.

By “reduce” or “inhibit” is meant the ability to cause an overalldecrease, for example, of 20% or greater, of 50% or greater, or of 75%,85%, 90%, 95%, or greater. In certain embodiments, reduce or inhibit canrefer to the effector function of an antibody that is mediated by theantibody Fc region, such effector functions specifically includingcomplement-dependent cytotoxicity (CDC), antibody-dependent cellularcytotoxicity (ADCC), and antibody-dependent cellular phagocytosis(ADCP).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).)A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an anti-CD3 antibody of the invention or a nucleicacid encoding an anti-CD3 antibody of the invention) or a composition(e.g., a pharmaceutical composition, e.g., a pharmaceutical compositionincluding an anti-CD3 antibody of the invention) to a subject. Thecompositions utilized in the methods described herein can beadministered, for example, intramuscularly, intravenously,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularly, orally, topically,locally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. The method ofadministration can vary depending on various factors (e.g., the compoundor composition being administered and the severity of the condition,disease, or disorder being treated).

II. Compositions and Methods

In one aspect, the invention is based, in part, on anti-CD3 antibodies.In certain embodiments, the anti-CD3 antibodies are multispecific (e.g.,bispecific) and bind, in addition to CD3 or a fragment thereof, a secondbiological molecule (e.g., a cell surface antigen, e.g., a tumorantigen). Antibodies of the invention are useful, for example, fortreating or delaying the progression of a cell proliferative disorder(e.g., cancer) or an autoimmune disorder, or for enhancing immunefunction in a subject having such a disorder.

A. Affinity Improved Anti-CD3 Antibodies

In one aspect, the invention provides isolated antibodies that bind toCD3 (e.g., CD3ε and/or CD3γ). In some instances the anti-CD3 antibodybinds to a human CD3 polypeptide or a cynomolgus monkey (cyno) CD3polypeptide. In some instances, the human CD3 polypeptide or the cynoCD3 polypeptide is a human CD3ε polypeptide (SEQ ID NO: 73) or a cynoCD3ε polypeptide (SEQ ID NO: 74), respectively. In some instances, thehuman CD3 polypeptide or the cyno CD3 polypeptide is a human CD3γpolypeptide (SEQ ID NO: 75) or a cyno CD3γ polypeptide (SEQ ID NO: 76),respectively. In some instances, the anti-CD3 antibody binds to anepitope within a fragment of CD3 (e.g., human CD3ε) consisting of aminoacids 1-26 (SEQ ID NO: 77) or 1-27 (SEQ ID NO: 78) of human CD3ε.

In some instances, the CD3 binding domain binds to an epitope consistingof amino acids of human CD3ε selected from GIn1, Asp2, Asn4, Glu6, andMet7. In one particular embodiment, the CD3 binding domain binds to anepitope that specifically includes Glu6. In certain other embodiments,the CD3 binding domain is provided that does not bind to an epitope thatincludes human CD3ε amino acid Glu5. In certain other embodiments, theCD3 binding domain is provided that does not bind to an epitope thatincludes human CD3ε amino acids Gly3 and Glu5.

A CD3 epitope may be determined by the CD3 binding domain binding topeptide fragments of the epitope. Alternatively, a CD3 epitope may bedetermined by alanine scanning mutagenesis. In one embodiment, areduction in binding of a CD3 binding domain to mutated CD3 by 20%, 30%,50%, 80% or more indicates the amino acid residue of CD3 mutated in analanine scanning mutagenesis assay is an epitope residue for that CD3binding domain. Alternatively, a CD3 epitope may be determined by massspectrometry. In some embodiments, the epitope is determined bycrystallography (e.g., crystallography methods). In some embodiments,the CD3 epitope as determined by crystallography is determined usingamino acids Q1-M7 of CD3. In some embodiments, the CD3 epitope asdetermined by crystallography is determined using amino acidsQDGNEEMGGITQTPYK (SEQ ID NO: 79) of CD3.

In some embodiments, the CD3 epitope as determined by crystallographymay be performed by combining the anti-CD3 antibody Fab, dissolved in0.15 M NaCl, 25 mM tris, pH 7.5 at 10 mg/ml, with a 2-fold molar excess(1 mg) of CD3ε peptide and initially screening a sparse matrix ofprecipitants in a sitting drop vapor diffusion format. Optimizedcrystals may be grown from a 1:1 mixture with reservoir solutioncontaining 70% v/v methyl-pentanediol, and 0.1 M HEPES buffer at pH 7.5.The reservoir may be used as a cryoprotectant. The crystals may betransferred to cryogenic temperature by sudden immersion into liquidnitrogen.

The diffraction data for crystals may be collected at Advanced PhotonSource beam line 221D, using a MAR300 CCD detector. The recordeddiffractions may be integrated and scaled using the program HKL2000.

The structure may be phased by molecular replacement (MR) method usingprogram Phaser. For example, the MR search model is a Fab subunitderived from a crystal structure of HGFA/Fab complex (PDB code: 2ROL).The CD3ε peptide is built into the structure based on a Fo-Fc map. Thestructure may be subsequently refined with programs REFMACS and PHENIXusing the maximum likelihood target functions, anisotropic individualB-factor refinement method, and TLS refinement method, to achieveconvergence.

In some instances, the invention provides an anti-CD3 antibody having abinding domain comprising at least an HVR-L3 sequence of TQSFILRT (SEQID NO: 6) and one, two, three, four, or five HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; and (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 5.

In some instances, the invention provides an anti-CD3 antibody having abinding domain comprising at least one, two, three, four, five, or sixHVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 6. In some instances,the anti-CD3 antibody may have a VH domain comprising an amino acidsequence having at least 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 7 and/or a VL domain comprising an amino acidsequence having at least 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 8. In a particular instance, the anti-CD3antibody can be 38E4v11, or a derivative or clonal relative thereof.

In some instances, an anti-CD3 antibody may comprise at least one (e.g.,1, 2, 3, or 4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, andFR-H4 comprising the sequences of SEQ ID NOs: 9-12, respectively, and/orat least one (e.g., 1, 2, 3, or 4) of the light chain framework regionsFR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs:13-16, respectively.

In any of the above embodiments, an anti-CD3 antibody is humanized. Inone embodiment, an anti-CD3 antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g., a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-CD3 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above, wherein one or both ofthe variable domain sequences include post-translational modifications.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-CD3 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD3 antibody comprising a VH sequence of SEQ ID NO: 7and a VL sequence of SEQ ID NO: 8. In certain embodiments, an antibodyis provided that binds to an epitope within a fragment of CD3 (e.g.,human CD3ε) consisting of amino acids 1-26 (SEQ ID NO: 77) or 1-27 (SEQID NO: 78) of human CD3ε.

In a further aspect of the invention, an anti-CD3 antibody according toany of the above embodiments is a monoclonal antibody. In otherembodiments, the anti-CD3 antibody is a chimeric or human antibody. Inone embodiment, an anti-CD3 antibody is an antibody fragment, forexample, a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. In anotherembodiment, the antibody is a full-length antibody, e.g., an intact IgGantibody (e.g., an intact IgG1 antibody) or other antibody class orisotype as defined herein.

In a further aspect, an anti-CD3 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-8 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM(e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 μMor 26 μM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on), or k_(a))and dissociation rates (k_(off), or k_(d)) are calculated using a simpleone-to-one Langmuir binding model (BIACORE Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) is calculatedas the ratio k_(off)/k_(on). See, for example, Chen et al., J. Mol.Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶M⁻¹s⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al. Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs (or portions thereof), for example,are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Sci. USA, 103:3557-0.3562 (2006).Additional methods include those described, for example, in U.S. Pat.No. 7,189,826 (describing production of monoclonal human IgM antibodiesfrom hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology(Trioma technology) is also described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In any one of the above aspects, the anti-CD3 antibody provided hereinis a multispecific antibody, for example, a bispecific antibody.Multispecific antibodies are antibodies (e.g., monoclonal antibodies)that have binding specificities for at least two different sites. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of CD3 (e.g., CD3ε or CD3γ). In certain embodiments, one of thebinding specificities is for CD3 (e.g., CD3ε or CD3γ) and the other isfor any other antigen (e.g., a second biological molecule, e.g., a cellsurface antigen, e.g., a tumor antigen). Accordingly, a bispecificanti-CD3 antibody may have binding specificities for CD3 and a secondbiological molecule, such as a second biological molecule (e.g., a tumorantigen) listed in Table 1 and described herein and in U.S. Pub. No.2010/0111856.

TABLE 1 Tumor antigen targets of the bispecific anti-CD3 antibodies ofthe invention Ly6G6D CD79a ETBR IL13Ralpha2 M-CSF P2X5 SSX-2 0772P CD79bETV6- IL20Ra MCSP p53 SSX-4 AML1 fusion protein adipophillin Cdc27 EZH2Intestinal mdm-2 PAP STEAP1 carboxyl esterase AIM-2 CDK4 FcRH1 IRTA2 MDPPAX5 STEAP1 ALDH1A1 CDKN2A FcRH2 Kallikrein 4 ME1 PBF STEAP2 alpha- CEAFcRH5 KIF20A Melan-A/ PMEL17 survivin actinin-4 MART-1 alpha- CLL1FLT3-ITD KK-LC-1 Meloe pml- SYT- foetoprotein RARalpha SSX1 or - fusionSSX2 fusion protein protein Amphiregulin CLPP FN1 KM-HN-1 MMP-2 PRAMETAG-1 ARTC1 COA-1 G250/MN/ K-ras MMP-7 PRDX5 TAG-2 CAIX ASLG659 CPSFGAGE-1,2,8 LAGE-1 MPF PSCA Telomerase ASPHD1 CRIPTO GAGE-3,4,5,6,7 LDLR-MRP4 PSCA hlg TENB2 fucosyltransferase ASfusion protein B7-H4 Cw6GDNF-Ra1 Lengsin MSG783 PSMA TGF-betaRII BAFF-R CXCR5 GEDA LGR5 MUC1PTPRK TMEFF1 BAGE-1 CXORF61 GFRA1 LY64 MUC5AC RAB38/ TMEM118 NY-MEL-1BCLX (L) cyclin D1 glypican-3 Ly6E mucin RAGE-1 TMEM46 BCR- Cyclin-A1GnTVf CD20 MUM-1f RBAF600 TRAG-3 ABL fusion protein (b3a2) beta-catenindek-can fusion gp100/Pmel17 LY6K MUM-2 RET Triosephosphate proteinisomerase BING-4 DKK1 GPC3 LYPD1 MUM-3 RGS5 TRP-1/gp75 B-RAF DR1 GPNMBMAGE-Al Myosin RhoC TRP-2 class I Brevican DR13 GPR172A MAGE-A10 NA88-ARNF43 TRP2-INT2 CALCA E16 GPR19 MAGE-Al2 Napi2b RNF43 TrpM4 CASP-5 EDARGPR54 MAGE-A2 NCA RU2AS Tyrosinase CASP-8 EFTUD2 HAVCR1 MAGE-A3 neo-PAPSAGE tyrosinase CD19 Elongation HER2 MAGE-A4 NFYC secernin 1 VEGF factor2 CD21 ENAH (hMena) HER-2/neu MAGE-A6 N-ras Sema 5b WT1 CD22 EpCAMHERV-K-MEL MAGE-A9 NY-BR-1 SIRT2 XAGE- 1b/GAGED2a CD33 EphA3 HLA-DOBMAGE-C1 NY-ESO- SLC35D3 EGFR-T790M; 1/LAGE-2 CD45 EphB2R hsp70-2 MAGE-C2OA1 SNRPD1 BMPR1B CD70 Epiregulin IDO1 mammaglobin-A OGT SOX10 CD72 EGFRIGF2B3 MART2 OS-9 Sp17 EGFR-G719A EGFR-G719C; EGFR-G719S; EGFR-L858REGFR-S768I EGFR-L861Q

The bispecific anti-CD3 antibody (e.g., any one of the anti-CD3antibodies described above) may have binding specificities for CD3 and asecond biological molecule, such as a human leukocyte antigen(HLA)-peptide complex presented on the cell surface by MHC. Thebispecific anti-CD3 antibody (e.g., any one of the anti-CD3 antibodiesdescribed above) may have binding specificities for CD3 and a secondbiological molecule, such as the peptide of the HLA-peptide complex,which may be selected from the group consisting of Ly6G6D (lymphocyteantigen 6 complex, locus G6D; Ly6-D, MEGT; NP_067079.2); 0772P (CA125,MUC16; Genbank accession no. AF36148); adipophilin (perilipin-2, Adiposedifferentiation-related protein, ADRP, ADFP, MGC10598; NCBI ReferenceSequence: NP_001113.2); AIM-2 (Absent In Melanoma 2, PYHIN4,Interferon-Inducible Protein AIM2; NCBI Reference Sequence:NP_004824.1); ALDH1A1 (Aldehyde Dehydrogenase 1 Family, Member A1,ALDH1, PUMB1, Retinaldehyde Dehydrogenase 1, ALDC, ALDH-E1, ALHDII,RALDH 1, EC 1.2.1.36, ALDH11, HEL-9, HEL-S-53e, HEL12, RALDH1,Acetaldehyde Dehydrogenase 1, Aldehyde Dehydrogenase 1, Soluble,Aldehyde Dehydrogenase, Liver Cytosolic, ALDH Class 1, EpididymisLuminal Protein 12, Epididymis Luminal Protein 9, Epididymis SecretorySperm Binding Protein Li 53e, Retinal Dehydrogenase 1, RaIDH1, AldehydeDehydrogenase Family 1 Member A1, Aldehyde Dehydrogenase, Cytosolic, EC1.2.1; NCBI Reference Sequence: NP_000680.2); alpha-actinin-4 (ACTN4,Actinin, Alpha 4, FSGS1, Focal Segmental Glomerulosclerosis 1,Non-Muscle Alpha-Actinin 4, F-Actin Cross-Linking Protein, FSGS,ACTININ-4, Actinin Alpha4 Isoform, alpha-actinin-4; NCBI ReferenceSequence: NP_004915.2); alpha-fetoprotein (AFP, HPAFP, FETA,alpha-1-fetoprotein, alpha-fetoglobulin, Alpha-1-fetoprotein,Alpha-fetoglobulin, HP; GenBank: AAB58754.1); Amphiregulin (AREG, SDGF,Schwannoma-Derived Growth Factor, Colorectum Cell-Derived Growth Factor,AR, CRDGF; GenBank: AAA51781.1); ARTC1 (ART1, ADP-Ribosyltransferase 1,Mono(ADP-Ribosyl)Transferase 1, ADP-Ribosyltransferase C2 And C3Toxin-Like 1, ART2, CD296, RT6, ADP-Ribosyltransferase 2, GPI-LinkedNAD(P)(+)-Arginine ADP-Ribosyltransferase 1, EC 2.4.2.31, CD296 Antigen;NP); ASLG659; ASPHD1 (Aspartate Beta-Hydroxylase Domain Containing 1,Aspartate Beta-Hydroxylase Domain-Containing Protein 1, EC 1.14.11.-, EC1.14.11; GenBank: AAI44153.1); B7-H4 (VTCN1, V-Set Domain Containing TCell Activation Inhibitor 1, B7H4, B7 Superfamily Member 1, ImmuneCostimulatory Protein B7-H4, B7h.5, T-Cell Costimulatory Molecule B7x,B7S1, B7X, VCTN1, H4, B7 Family Member, PRO1291, B7 Family Member, H4, TCell Costimulatory Molecule B7x, V-Set Domain-Containing T-CellActivation Inhibitor 1, Protein B7S1; GenBank: AAZ17406.1); BAFF-R(TNFRSF13C, Tumor Necrosis Factor Receptor Superfamily, Member 13C,BAFFR, B-Cell-Activating Factor Receptor, BAFF Receptor, BLyS Receptor3, CVID4, BROMIX, CD268, B Cell-Activating Factor Receptor, prolixin,Tumor Necrosis Factor Receptor Superfamily Member 13C, BR3, CD268Antigen; NCBI Reference Sequence: NP_443177.1); BAGE-1; BCLX (L);BCR-ABL fusion protein (b3a2); beta-catenin (CTNNB1, Catenin(Cadherin-Associated Protein), Beta 1, 88kDa, CTNNB, MRD19, Catenin(Cadherin-Associated Protein), Beta 1 (88kD), armadillo, Catenin Beta-1;GenBank: CAA61107.1); BING-4 (WDR46, WD Repeat Domain 46, C6orf11,BING4, WD Repeat-Containing Protein BING4, Chromosome 6 Open ReadingFrame 11, FP221, UTP7, WD Repeat-Containing Protein 46; NP); BMPR1B(bone morphogenetic protein receptor-type IB, Genbank accession no.NM_00120; NP); B-RAF (Brevican (BCAN, BEHAB, Genbank accession no.AF22905); Brevican (BCAN, Chondroitin Sulfate Proteoglycan 7,Brain-Enriched Hyaluronan-Binding Protein, BEHAB, CSPG7, BrevicanProteoglycan, Brevican Core Protein, Chondroitin Sulfate ProteoglycanBEHAB; GenBank: AAH27971.1); CALCA (Calcitonin-Related PolypeptideAlpha, CALC1, Calcitonin 1, calcitonin, Alpha-Type CGRP, CalcitoninGene-Related Peptide I, CGRP-I, CGRP, CGRP1, CT, KC,Calcitonin/Calcitonin-Related Polypeptide, Alpha, katacalcin; NP);CASP-5 (CASPS, Caspase 5, Apoptosis-Related Cysteine Peptidase, Caspase5, Apoptosis-Related Cysteine Protease, Protease ICH-3, Protease TY,ICE(rel)-III, ICE(rel)III, ICEREL-Ill, ICH-3, caspase-5, TY Protease, EC3.4.22.58, ICH3, EC 3.4.22; NP); CASP-8; CD19 (CD19—B-lymphocyte antigenCD19 isoform 2 precursor, B4, CVID3 [Homo sapiens], NCBI ReferenceSequence: NP_001761.3); CD20 (CD20—B-lymphocyte antigen CD20,membrane-spanning 4-domains, subfamily A, member 1, B1, Bp35, CD20,CVID5, LEU-16, MS4A2, S7; NCBI Reference Sequence: NP_690605.1); CD21(CD21 (CR2 (Complement receptor or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M2600); (CD22 (B-cellreceptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814,Genbank accession No. AK02646); CD22; CD33 (CD33 Molecule, CD33 Antigen(Gp67), Sialic Acid Binding Ig-Like Lectin 3, Sialic Acid-BindingIg-Like Lectin 3, SIGLEC3, gp67, SIGLEC-3, Myeloid Cell Surface AntigenCD33, p67, Siglec-3, CD33 Antigen; GenBank: AAH28152.1); CD45; CD70(CD70—tumor necrosis factor (ligand) superfamily, member 7; surfaceantigen CD70; Ki-24 antigen; CD27 ligand; CD27-L; tumor necrosis factorligand superfamily member 7; NCBI Reference Sequence for species Homosapiens: NP_001243.1); CD72 (CD72 (B-cell differentiation antigen CD72,Lyb-; 359 aa, pl: 8.66, MW: 40225, TM: 1 [P] Gene Chromosome: 9p13.3,Genbank accession No. NP_001773.); CD79a (CD79a (CD79A, CD79a,immunoglobulin-associated alpha, a B cell-specific protein thatcovalently interacts with Ig beta (CD79B) and forms a complex on thesurface with Ig M molecules, transduces a signal involved in B-celldifferentiation), pl: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome:19q13.2, Genbank accession No. NP_001774.1); CD79b (CD79b (CD79B, CD79b,IGb (immunoglobulin-associated beta), B29, Genbank accession no.NM_000626 or 1103867); Cdc27 (Cell Division Cycle 27, D0S1430E,D175978E, Anaphase Promoting Complex Subunit 3, Anaphase-PromotingComplex Subunit 3, ANAPC3, APC3, CDC27Hs, H-NUC, CDC27 Homolog, CellDivision Cycle 27 Homolog (S. cerevisiae), HNUC, NUC2,Anaphase-Promoting Complex, Protein 3, Cell Division Cycle 27 Homolog,Cell Division Cycle Protein 27 Homolog, Nuc2 Homolog; GenBank:AAH11656.1); CDK4 (Cyclin-Dependent Kinase 4, Cell Division ProteinKinase 4, PSK-J3, EC 2.7.11.22, CMM3, EC 2.7.11; NCBI ReferenceSequence: NP_000066.1); CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A,MLM, CDKN2, MTS1, Cyclin-Dependent Kinase Inhibitor 2A (Melanoma, P16,Inhibits CDK4), Cyclin-Dependent Kinase 4 Inhibitor A, Multiple TumorSuppressor 1, CDK4I, MTS-1, CMM2, P16, ARF, INK4, INK4A, P14, P14ARF,P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16, CDK4 InhibitorP16-INK4, Cell Cycle Negative Regulator Beta, p14ARF, p16-INK4,p16-INK4a, p16INK4A, p19ARF; NP); CEA; CLL1 (CLL-1 (CLEC12A, MICL, andDCAL, encodes a member of the C-type lectin/C-type lectin-like domain(CTL/CTLD) superfamily. Members of this family share a common proteinfold and have diverse functions, such as cell adhesion, cell-cellsignaling, glycoprotein turnover, and roles in inflammation and immuneresponse. The protein encoded by this gene is a negative regulator ofgranulocyte and monocyte function. Several alternatively splicedtranscript variants of this gene have been described, but thefull-length nature of some of these variants has not been determined.This gene is closely linked to other CTL/CTLD superfamily members in thenatural killer gene complex region on chromosome 12p13 (Drickamer KCurr. Opin. Struct. Biol. 9):585-90; van Rhenen A, et al., Blood110):2659-66; Chen C H, et al. Blood 107):1459-67; Marshall A S, et al.Eur. J. Immunol. 36):2159-69; Bakker A B, et al Cancer Res. 64:8443-50;Marshall A S, et al J. Biol. Chem. 279:14792-80. CLL-1 has been shown tobe a type II transmembrane receptor comprising a single C-typelectin-like domain (which is not predicted to bind either calcium orsugar), a stalk region, a transmembrane domain and a short cytoplasmictail containing an ITIM motif.); CLPP (Caseinolytic Mitochondrial MatrixPeptidase Proteolytic Subunit, Endopeptidase Clp, EC 3.4.21.92, PRLTS3,ATP-Dependent Protease CIpAP (E. coli), CIpP (Caseinolytic Protease,ATP-Dependent, Proteolytic Subunit, E. coli) Homolog, CIpP CaseinolyticPeptidase, ATP-Dependent, Proteolytic Subunit Homolog (E. coli), CIpPCaseinolytic Protease, ATP-Dependent, Proteolytic Subunit Homolog (E.coli), human, Proteolytic Subunit, ATP-Dependent Protease CIpAP,Proteolytic Subunit, Human, CIpP Caseinolytic Peptidase ATP-Dependent,Proteolytic Subunit, CIpP Caseinolytic Peptidase, ATP-Dependent,Proteolytic Subunit Homolog, CIpP Caseinolytic Protease, ATP-Dependent,Proteolytic Subunit Homolog, Putative ATP-Dependent Clp ProteaseProteolytic Subunit, Mitochondrial; NP); COA-1; CPSF; CRIPTO (CRIPTO(CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor,Genbank accession no. NP_003203 or NM_00321); Cw6; CXCR5 CXCR5(Burkitt's lymphoma receptor 1, a G protein-coupled receptor that isactivated by the CXCL13 chemokine, functions in lymphocyte migration andhumoral defense, plays a role in HIV-2 infection and perhaps developmentof AIDS, lymphoma, myeloma, and leukemia); 372 aa, pl: 8.54 MW: 41959TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession No. NP_001707.);CXORF61 CXORF61—chromosome X open reading frame 61 [Homo sapiens], NCBIReference Sequence: NP_001017978.1); cyclin D1 (CCND1, BCL1, PRAD1,D11S287E, B-Cell CLL/Lymphoma 1, B-Cell Lymphoma 1 Protein, BCL-1Oncogene, PRAD1 Oncogene, Cyclin D1 (PRAD1: Parathyroid Adenomatosis 1),G1/S-Specific Cyclin D1, Parathyroid Adenomatosis 1, U21B31,G1/S-Specific Cyclin-D1, BCL-1; NCBI Reference Sequence: NP_444284.1);Cyclin-A1 (CCNA1, CT146, Cyclin A1; GenBank: AAH36346.1); dek-can fusionprotein; DKK1 (Dickkopf WNT Signaling Pathway Inhibitor 1, SK, hDkk-1,Dickkopf (Xenopus Laevis) Homolog 1, Dickkopf 1 Homolog (XenopusLaevis), DKK-1, Dickkopf 1 Homolog, Dickkopf Related Protein-1,Dickkopf-1 Like, Dickkopf-Like Protein 1, Dickkopf-Related Protein 1,Dickkopf-1, Dkk-1; GenBank: AAQ89364.1); DR1 (Down-Regulator OfTranscription 1, TBP-Binding (Negative Cofactor 2), Negative Cofactor2-Beta, TATA-Binding Protein-Associated Phosphoprotein, NC2, NC2-BETA,Protein Dr1, NC2-beta, Down-Regulator Of Transcription 1; NCBI ReferenceSequence: NP_001929.1); DR13 (Major Histocompatibility Complex, ClassII, DR Beta 1, HLA-DR1B, DRw10, DW2.2/DR2.2, SS1, DRB1, HLA-DRB, HLAClass II Histocompatibility Antigen, DR-1 Beta Chain, Human LeucocyteAntigen DRB1, Lymphocyte Antigen DRB1, MHC Class II Antigen, MHC ClassII HLA-DR Beta 1 Chain, MHC Class II HLA-DR-Beta Cell SurfaceGlycoprotein, MHC Class II HLA-DRw10-Beta, DR-1, DR-12, DR-13, DR-14,DR-16, DR-4, DR-5, DR-7, DR-8, DR-9, DR1, DR12, DR13, DR14, DR16, DR4,DRS, DR7, DRB, DR9, DRw11, DRw8, HLA-DRB2, Clone P2-Beta-3, MHC Class IIAntigen DRB1*1, MHC Class II Antigen DRB1*10, MHC Class II AntigenDRB1*11, MHC Class II Antigen DRB1*12, MHC Class II Antigen DRB1*13, MHCClass II Antigen DRB1*14, MHC Class II Antigen DRB1*15, MHC Class IIAntigen DRB1*16, MHC Class II Antigen DRB1*3, MHC Class II AntigenDRB1*4, MHC Class II Antigen DRB1*7, MHC Class II Antigen DRB1*8, MHCClass II Antigen DRB1*9; NP); E16 (E16 (LAT1, SLC7A5, Genbank accessionno. NM_00348); EDAR (EDAR—tumor necrosis factor receptor superfamilymember EDAR precursor, EDA-A1 receptor; downless homolog;ectodysplasin-A receptor; ectodermal dysplasia receptor; anhidroticectodysplasin receptor 1, DL; ECTD10A; ECTD10B; ED1R; ED3; EDS; EDA-A1R;EDA1R; EDA3; HRM1 [Homo sapiens]; NCBI Reference Sequence: NP_071731.1);EFTUD2 (Elongation Factor Tu GTP Binding Domain Containing 2, ElongationFactor Tu GTP-Binding Domain-Containing Protein 2, hSNU114, SNU114Homolog, U5 SnRNP-Specific Protein, 116 KDa, MFDGA, KIAA0031, 116 KD, U5SnRNP Specific Protein, 116 KDa U5 Small Nuclear RibonucleoproteinComponent, MFDM, SNRNP116, Snrp116, Snu114, U5-116KD, SNRP116, U5-116KDa; GenBank: AAH02360.1); EGFR (Epidermal Growth Factor Receptor, ERBB,Proto-Oncogene C-ErbB-1, Receptor Tyrosine-Protein Kinase ErbB-1, ERBB1,HER1, EC 2.7.10.1, Epidermal Growth Factor Receptor (AvianErythroblastic Leukemia Viral (V-Erb-B) Oncogene Homolog),Erythroblastic Leukemia Viral (V-Erb-B) Oncogene Homolog (Avian), PIG61,Avian Erythroblastic Leukemia Viral (V-Erb-B) Oncogene Homolog, CellGrowth Inhibiting Protein 40, Cell Proliferation-Inducing Protein 61,mENA, EC 2.7.10; GenBank: AAH94761.1); EGFR-G719A; EGFR-G719C;EGFR-G719S; EGFR-L858R; EGFR-L861Q; EGFR-S7681; EGFR-T790M; Elongationfactor 2 (EEF2, Eukaryotic Translation Elongation Factor 2, EF2,Polypeptidyl-TRNA Translocase, EF-2, SCA26, EEF-2; NCBI ReferenceSequence: NP_001952.1); ENAH (hMena) (Enabled Homolog (Drosophila),MENA, Mammalian Enabled, ENA, NDPP1, Protein Enabled Homolog; GenBank:AAH95481.1)—results for just “ENAH” not “ENAH (hMena)”; EpCAM(Epithelial Cell Adhesion Molecule, M4S1, MIC18, Tumor-AssociatedCalcium Signal Transducer 1, TACSTD1, TROP1, Adenocarcinoma-AssociatedAntigen, Cell Surface Glycoprotein Trop-1, Epithelial Glycoprotein 314,Major Gastrointestinal Tumor-Associated Protein GA733-2, EGP314, KSA,DIAR5, HNPCC8, Antigen Identified By Monoclonal Antibody AUA1, EGP-2,EGP40, ESA, KS1/4, MK-1, Human Epithelial Glycoprotein-2, MembraneComponent, Chromosome 4, Surface Marker (35kD Glycoprotein), EGP,Ep-CAM, GA733-2, M1S2, CD326 Antigen, Epithelial Cell Surface Antigen,hEGP314, KS 1/4 Antigen, ACSTD1; GenBank: AAH14785.1); EphA3 (EPHReceptor A3, ETK1, ETK, TYRO4, HEK, Eph-Like Tyrosine Kinase 1,Tyrosine-Protein Kinase Receptor ETK1, EK4, EPH-Like Kinase 4, EC2.7.10.1, EPHA3, HEK4, Ephrin Type-A Receptor 3, Human Embryo Kinase 1,TYRO4 Protein Tyrosine Kinase, hEK4, Human Embryo Kinase,Tyrosine-Protein Kinase TYRO4, EC 2.7.10; GenBank: AAH63282.1); EphB2R;Epiregulin (EREG, ER, proepiregulin; GenBank: AA136405.1); ETBR (EDNRB,Endothelin Receptor Type B, HSCR2, HSCR, Endothelin ReceptorNon-Selective Type, ET-B, ET-BR, ETRB, ABCDS, WS4A, ETB, Endothelin BReceptor; NP); ETV6-AML1 fusion protein; EZH2 (Enhancer Of Zeste Homolog2 (Drosophila), Lysine N-Methyltransferase 6, ENX-1, KMT6 EC 2.1.1.43,EZH1, WVS, Enhancer Of Zeste (Drosophila) Homolog 2, ENX1, EZH2b, KMT6A,WVS2, Histone-Lysine N-Methyltransferase EZH2, Enhancer Of Zeste Homolog2, EC 2.1.1; GenBank: AAH10858.1); FcRH1 (FCRL1, Fc Receptor-Like 1,FCRH1, Fc Receptor Homolog 1, FcR-Like Protein 1, Immune ReceptorTranslocation-Associated Protein 5, IFGP1, IRTA5, hIFGP1, IFGP FamilyProtein 1, CD307a, Fc Receptor-Like Protein 1, ImmunoglobulinSuperfamily Fc Receptor, Gp42, FcRL1, CD307a Antigen; GenBank:AAH33690.1); FcRH2 (FCRL2, Fc Receptor-Like 2, SPAP1, SH2Domain-Containing Phosphatase Anchor Protein 1, Fc Receptor Homolog 2,FcR-Like Protein 2, Immunoglobulin Receptor Translocation-AssociatedProtein 4, FCRH2, IFGP4, IRTA4, IFGP Family Protein 4, SPAP1A, SPAP1B,SPAP1C, CD307b, Fc Receptor-Like Protein 2, Immune ReceptorTranslocation-Associated Protein 4, Immunoglobulin Superfamily FcReceptor, Gp42, SH2 Domain Containing Phosphatase Anchor Protein 1,FcRL2, CD307b Antigen; Gen Bank: AAQ88497.1); FcRH5 (FCRLS, FcReceptor-Like 5, IRTA2, Fc Receptor Homolog 5, FcR-Like Protein 5,Immune Receptor Translocation-Associated Protein 2, BXMAS1, FCRHS,CD307, CD307e, PRO820, Fc Receptor-Like Protein 5, ImmunoglobulinSuperfamily Receptor Translocation Associated 2 (IRTA2), FcRL5, CD307eAntigen; GenBank: AAI01070.1); FLT3-ITD; FN1(Fibronectin 1,Cold-Insoluble Globulin, FN, Migration-Stimulating Factor, CIG, FNZ,GFND2, LETS, ED-B, FINC, GFND, MSF, fibronectin; GenBank: AA143764.1);G250 (MN, CAIX, Carbonic Anhydrase IX, Carbonic Dehydratase,RCC-Associated Protein G250, Carbonate Dehydratase IX, Membrane AntigenMN, Renal Cell Carcinoma-Associated Antigen G250, CA-IX, P54/58N, pMW1,RCC-Associated Antigen G250, Carbonic Anhydrase 9; NP);—alias resultsfor “G250” not “G250/MN/CAIX”; GAGE-1,2,8; GAGE-3,4,5,6,7; GDNF-Ra1(GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1;RET1L; GDNFR-alpha1; GFR-ALPHA-; U95847; BC014962; NM_145793 NM_005264);GEDA (Genbank accession No. AY26076); GFRA1—GDNF family receptoralpha-1; GDNF receptor alpha-1; GDNFR-alpha-1; GFR-alpha-1; RET ligand1; TGF-beta-related neurotrophic factor receptor 1 [Homo sapiens];ProtKB/Swiss-Prot: P56159.2; glypican-3 (GPC3, Glypican 3, SDYS,Glypican Proteoglycan 3, Intestinal Protein OCI-5, GTR2-2, MXR7, SGBS1,DGSX, OCI-5. SGB, SGBS, Heparan Sulphate Proteoglycan, SecretedGlypican-3, 0015; GenBank: AAH35972.1); GnTVf; gp100 (PMEL,Premelanosome Protein, SILV, D12S53E, PMEL17, SIL, Melanocyte ProteinPmel 17, Melanocytes Lineage-Specific Antigen GP100, Melanoma-AssociatedME20 Antigen, Silver Locus Protein Homolog, ME20-M, ME20M, P1, P100,Silver (Mouse Homolog) Like, Silver Homolog (Mouse), ME20, SI,Melanocyte Protein Mel 17, Melanocyte Protein PMEL, Melanosomal MatrixProtein17, Silver, Mouse, Homolog Of; GenBank: AAC60634.1); GPC; GPNMB(Glycoprotein (Transmembrane) Nmb, Glycoprotein NMB, GlycoproteinNmb-Like Protein, osteoactivin, Transmembrane Glycoprotein HGFIN, HGFIN,NMB, Transmembrane Glycoprotein, Transmembrane Glycoprotein NMB;GenBank: AAH32783.1); GPR172A (G protein-coupled receptor 172A; GPCR41;FLJ11856; D15Ertd747e); NP_078807.1; NM_024531.3); GPR19 (Gprotein-coupled receptor 19; Mm.478; NP_006134.1; NM_006143.2); GPR54(KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR1; NP_115940.2;NM_032551.4); HAVCR1 (Hepatitis A Virus Cellular Receptor 1, T-CellImmunoglobulin Mucin Family Member 1, Kidney Injury Molecule 1, KIM-1,KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1, T-Cell Immunoglobulin MucinReceptor 1, T-Cell Membrane Protein 1, HAVCR, HAVCR-1, T CellImmunoglobin Domain And Mucin Domain Protein 1, HAVcr-1, T-CellImmunoglobulin And Mucin Domain-Containing Protein 1; GenBank:AAH13325.1); HER2 (ERBB2, V-Erb-B2 Avian Erythroblastic Leukemia ViralOncogene Homolog 2, NGL, NEU, Neuro/Glioblastoma Derived OncogeneHomolog, Metastatic Lymph Node Gene 19 Protein, Proto-Oncogene C-ErbB-2,Proto-Oncogene Neu, Tyrosine Kinase-Type Cell Surface Receptor HER2, MLN19, p185erbB2, EC 2.7.10.1, V-Erb-B2 Avian Erythroblastic Leukemia ViralOncogene Homolog 2 (Neuro/Glioblastoma Derived Oncogene Homolog), CD340,HER-2, HER-2/neu, TKR1, C-Erb B2/Neu Protein, herstatin,Neuroblastoma/Glioblastoma Derived Oncogene Homolog, ReceptorTyrosine-Protein Kinase ErbB-2, V-Erb-B2 Erythroblastic Leukemia ViralOncogene Homolog 2, Neuro/Glioblastoma Derived Oncogene Homolog, MLN19,CD340 Antigen, EC 2.7.10; NP); HER-2/neu—alias of above; HERV-K-MEL;HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that bindspeptides and presents them to CD4+T lymphocytes); 273 aa, p1: 6.56, MW:30820.TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_002111); hsp70-2 (HSPA2, Heat Shock 70kDa Protein 2, Heat Shock 70kDProtein 2, HSP70-3, Heat Shock-Related 70 KDa Protein 2, Heat Shock 70KDa Protein 2; GenBank: AAD21815.1); IDO1 (Indoleamine 2,3-Dioxygenase1, IDO, INDO, Indoleamine-Pyrrole 2,3-Dioxygenase, IDO-1,Indoleamine-Pyrrole 2,3 Dioxygenase, Indolamine 2,3 Dioxygenase, Indole2,3 Dioxygenase, EC 1.13.11.52; NCBI Reference Sequence: NP_002155.1);IGF2B3; IL13Ralpha2 (IL13RA2, Interleukin 13 Receptor, Alpha 2,Cancer/Testis Antigen 19, Interleukin-13-Binding Protein,IL-13R-alpha-2, IL-13RA2, IL-13 Receptor Subunit Alpha-2, IL-13R SubunitAlpha-2, CD213A2, CT19, IL-13R, IL13BP, Interleukin 13 Binding Protein,Interleukin 13 Receptor Alpha 2 Chain, Interleukin-13 Receptor SubunitAlpha-2, IL13R, CD213a2 Antigen; NP); IL20Rα; Intestinal carboxylesterase; IRTA2 (alias of FcRH5); Kallikrein 4 (KLK4, Kallikrein-RelatedPeptidase 4, PRSS17, EMSP1, Enamel Matrix Serine Proteinase 1,Kallikrein-Like Protein 1, Serine Protease 17, KLK-L1, PSTS, AI2A1,Kallikrein 4 (Prostase, Enamel Matrix, Prostate), ARM1, EMSP,Androgen-Regulated Message 1, Enamel Matrix Serine Protease 1,kallikrein, kallikrein-4, prostase, EC 3.4.21.-, Prostase, EC 3.4.21;GenBank: AAX30051.1); KIF20A (Kinesin Family Member 20A, RAB6KIFL, RAB6Interacting, Kinesin-Like (Rabkinesin6), Mitotic a; LAGE-1;LDLR-fucosyltransferaseASfusion protein; Lengsin (LGSN, Lengsin, LensProtein With Glutamine Synthetase Domain, GLULD1, Glutamate-AmmoniaLigase Domain-Containing Protein 1, LGS, Glutamate-Ammonia Ligase(Glutamine Synthetase) Domain Containing 1, Glutamate-Ammonia Ligase(Glutamine Synthase) Domain Containing 1, Lens Glutamine Synthase-Like;GenBank: AAF61255.1); LGR5 (leucine-rich repeat-containing Gprotein-coupled receptor 5; GPR49, GPR6; NP_003658.1; NM_003667.2; LY64(Lymphocyte antigen 64 (RP10, type I membrane protein of the leucinerich repeat (LRR) family, regulates B-cell activation and apoptosis,loss of function is associated with increased disease activity inpatients with systemic lupus erythematosis); 661 aa, pl: 6.20, MW: 74147TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No. NP_005573.; Ly6E(lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-;NP_002337.1; NM_002346.2); LY6K (lymphocyte antigen 6 complex, locus K;LY6K; HSJ001348; FLJ3522; NP_059997.3; NM_017527.3); LyPD1-LY6/PLAURdomain containing 1, PHTS [Homo sapiens], GenBank: AAH17318.1); MAGE-A1(Melanoma Antigen Family A, 1 (Directs Expression Of Antigen MZ2-E,MAGE1, Melanoma Antigen Family A 1, MAGEA1, Melanoma Antigen MAGE-1,Melanoma-Associated Antigen 1, Melanoma-Associated Antigen MZ2-E,Antigen MZ2-E, Cancer/Testis Antigen 1.1, CT1.1, MAGE-1 Antigen,Cancer/Testis Antigen Family 1, Member 1, Cancer/Testis Antigen Family1, Member 1, MAGE1A; NCBI Reference Sequence: NP_004979.3); MAGE-A10(MAGEA10, Melanoma Antigen Family A, 10, MAGE10, MAGE-10 Antigen,Melanoma-Associated Antigen 10, Cancer/Testis Antigen 1.10, CT1.10,Cancer/Testis Antigen Family 1, Member 10, Cancer/Testis Antigen Family1, Member 10; NCBI Reference Sequence: NP_001238757.1); MAGE-A12(MAGEA12, Melanoma Antigen Family A, 12, MAGE12, Cancer/Testis Antigen1.12, CT1.12, MAGE12F Antigen, Cancer/Testis Antigen Family 1, Member12, Cancer/Testis Antigen Family 1, Member 12, Melanoma-AssociatedAntigen 12, MAGE-12 Antigen; NCBI Reference Sequence: NP_001159859.1);MAGE-A2 (MAGEA2, Melanoma Antigen Family A, 2, MAGE2, Cancer/TestisAntigen 1.2, CT1.2, MAGEA2A, MAGE-2 Antigen, Cancer/Testis AntigenFamily 1, Member 2, Cancer/Testis Antigen Family 1, Member 2, MelanomaAntigen 2, Melanoma-Associated Antigen 2; NCBI Reference Sequence:NP_001269434.1); MAGE-A3 (MAGEA3, Melanoma Antigen Family A, 3, MAGE3,MAGE-3 Antigen, Antigen MZ2-D, Melanoma-Associated Antigen 3,Cancer/Testis Antigen 1.3, CT1.3, Cancer/Testis Antigen Family 1, Member3, HIPS, HYPD, MAGEA6, Cancer/Testis Antigen Family 1, Member 3; NCBIReference Sequence: NP_005353.1); MAGE-A4 (MAGEA4, Melanoma AntigenFamily A, 4, MAGE4, Melanoma-Associated Antigen 4, Cancer/Testis Antigen1.4, CT1.4, MAGE-4 Antigen, MAGE-41 Antigen, MAGE-X2 Antigen, MAGE4A,MAGE4B, Cancer/Testis Antigen Family 1, Member 4, MAGE-41, MAGE-X2,Cancer/Testis Antigen Family 1, Member 4; NCBI Reference Sequence:NP_001011550.1); MAGE-A6 (MAGEA6, Melanoma Antigen Family A, 6, MAGE6,MAGE-6 Antigen, Melanoma-Associated Antigen 6, Cancer/Testis Antigen1.6, CT1.6, MAGE3B Antigen, Cancer/Testis Antigen Family 1, MelanomaAntigen Family A 6, Member 6, MAGE-3b, MAGE3B, Cancer/Testis AntigenFamily 1, Member 6; NCBI Reference Sequence: NP_787064.1); MAGE-A9(MAGEA9, Melanoma Antigen Family A, 9, MAGE9, MAGE-9 Antigen,Melanoma-Associated Antigen 9, Cancer/Testis Antigen 1.9, CT1.9,Cancer/Testis Antigen Family 1, Member 9, Cancer/Testis Antigen Family1, Member 9, MAGEA9A; NCBI Reference Sequence: NP_005356.1); MAGE-C1(MAGEC1, Melanoma Antigen Family C, 1, Cancer/Testis Antigen 7.1, CT7.1,MAGE-C1 Antigen, Cancer/Testis Antigen Family 7, Member 1, CT7,Cancer/Testis Antigen Family 7, Member 1, Melanoma-Associated AntigenC1; NCBI Reference Sequence: NP_005453.2); MAGE-C2 (MAGEC2, MelanomaAntigen Family C, 2, MAGEE1, Cancer/Testis Antigen 10, CT10, HCA587,Melanoma Antigen, Family E, 1, Cancer/Testis Specific, HepatocellularCarcinoma-Associated Antigen 587, MAGE-C2 Antigen, MAGE-E1 Antigen,Hepatocellular Cancer Antigen 587, Melanoma-Associated Antigen C2; NCBIReference Sequence: NP_057333.1); mammaglobin-A (SCGB2A2, Secretoglobin,Family 2A, Member 2, MGB1, Mammaglobin 1, UGB2, Mammaglobin A,mammaglobin-A, Mammaglobin-1, Secretoglobin Family 2A Member 2; NP);MART2 (HHAT, Hedgehog Acyltransferase, SKI1, Melanoma Antigen RecognizedBy T-Cells 2, Skinny Hedgehog Protein 1, Skn, Melanoma AntigenRecognized By T Cells 2, Protein-Cysteine N-Palmitoyltransferase HHAT,EC 2.3.1.-; GenBank: AAH39071.1); M-CSF (CSF1, Colony Stimulating Factor1 (Macrophage), MCSF, CSF-1, lanimostim, Macrophage Colony-StimulatingFactor 1, Lanimostim; GenBank: AAH21117.1); MCSP (SMCP, SpermMitochondria-Associated Cysteine-Rich Protein, MCS, MitochondrialCapsule Selenoprotein, HSMCSGEN1, Sperm Mitochondrial-AssociatedCysteine-Rich Protein; NCBI Reference Sequence: NP_109588.2); XAGE-1b/GAGED2a; WT1 (Wilms Tumor 1, WAGR, GUD, WIT-2, WT33, Amino-TerminalDomain Of EWS, NPHS4, Last Three Zinc Fingers Of The DNA-Binding DomainOf WT1, AWT1, Wilms Tumor Protein, EWS-WT1; GenBank: AAB33443.1); VEGF;Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP; NP_000363.1;NM_000372.4; GenBank: AAB60319.1); TrpM4 (BR22450, FLJ20041, TRPM4,TRPM4B, transient receptor potential cation channel, subfamily M, member4, Genbank accession no. NM_01763); TRP2-INT2; TRP-2; TRP-1/gp75(Tyrosinase-Related Protein 1, 5,6-Dihydroxyindole-2-Carboxylic AcidOxidase, CAS2, CATB, TYRP, OCA3, Catalase B, b-PROTEIN, Glycoprotein 75,EC 1.14.18., Melanoma Antigen Gp75, TYRP1, TRP, TYRRP, TRP1, SHEP11,DHICA Oxidase, EC 1.14.18, GP75, EC 1.14.18.1; Triosephosphate isomerase(Triosephosphate isomerase 1, TPID, Triose-Phosphate Isomerase,HEL-S-49, TIM, Epididymis Secretory Protein Li 49, TPI, TriosephosphateIsomerase, EC 5.3.1.1; TRAG-3 (CSAG Family Member 2, Cancer/TestisAntigen Family 24, CSAG3B, Member 2, CSAG Family Member 3B,Cancer/Testis Antigen Family 24 Member 2, Cancer/Testis Antigen 24.2,Chondrosarcoma-Associated Gene 2/3 Protein, Taxol-Resistant-AssociatedGene 3 Protein, Chondrosarcoma-Associated Gene 2/3 Protein-Like, CT24.2,Taxol Resistance Associated Gene 3, TRAG-3, CSAG3A, TRAG3;); TMEM46(shisa homolog 2 (Xenopus laevis); SHISA; NP_001007539.1;NM_001007538.1; TMEM118 (ring finger protein, transmembrane2; RNFT2;FLJ1462; NP_001103373.1; NM_001109903.1; TMEFF1 (transmembrane proteinwith EGF-like and two follistatin-like domains 1; Tomoregulin-; H7365;C9orf2; C9ORF2; U19878; X83961; NM_080655; NM_003692; TGF-betaRII(TGFBR2, Transforming Growth Factor, Beta Receptor II (70/80kDa),TGFbeta-RII, MFS2, tbetaR-II, TGFR-2, TGF-Beta Receptor Type IIB,TGF-Beta Type II Receptor, TGF-Beta Receptor Type-2, EC 2.7.11.30,Transforming Growth Factor Beta Receptor Type IIC, AAT3, TbetaR-II,Transforming Growth Factor, Beta Receptor II (70-80kD), TGF-BetaReceptor Type II, FAA3, Transforming Growth Factor-Beta Receptor TypeII, LDS1B, HNPCC6, LDS2B, LDS2, RIIC, EC 2.7.11, TAAD2; TENB2 (TMEFF2,tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan,related to the EGF/heregulin family of growth factors and follistatin);374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq:NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbankaccession No. AF179274; AY358907, CAF85723, CQ782436; TAG-2; TAG-1(Contactin 2 (Axonal), TAG-1, AXT, Axonin-1 Cell Adhesion Molecule, TAX,Contactin 2 (transiently Expressed), TAX1, Contactin-2, AxonalGlycoprotein TAG-1, Transiently-Expressed Axonal Glycoprotein, TransientAxonal Glycoprotein, Axonin-1, TAX-1, TAG1, FAMES; PRF: 444868);SYT-SSX1 or -SSX2 fusion protein; survivin; STEAP2 (HGNC_8639, IPCA-1,PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1,prostate cancer associated protein 1, six transmembrane epithelialantigen of prostate 2, six transmembrane prostate protein, Genbankaccession no. AF45513; STEAP1 (six transmembrane epithelial antigen ofprostate, Genbank accession no. NM_01244; SSX-4; SSX-2 (SSX2, SynovialSarcoma, X Breakpoint2, X Breakpoint 2, SSX, X Breakpoint 2B,Cancer/Testis Antigen 5.2, X-Chromosome-Related 2, Tumor AntigenHOM-MEL-40, CT5.2, HD21, Cancer/Testis Antigen Family 5, HOM-MEL-40,Isoform B, Cancer/Testis Antigen Family 5 member 2a, member 2a, ProteinSSX2, Sarcoma, Sarcoma, Synovial, X-Chromosome-Related 2, synovial,Synovial Sarcoma, X Breakpoint 2B, Synovial Sarcomam, SSX2A; Sp17; SOX10(SRY (Sex Determining Region Y)-Box 10, mouse, PCWH, DOM, WS4, WS2E,WS4C, Dominant Megacolon, mouse, Human Homolog Of, Dominant Megacolon,SRY-Related HMG-Box Gene 10, Human Homolog Of, transcription FactorSOX-10; GenBank: CAG30470.1); SNRPD1 (Small Nuclear RibonucleoproteinD1, Small Nuclear Ribonucleoprotein D1, Polypeptide 16kDa, Polypeptide(16kD), SNRPD, HsT2456, Sm-D1, SMD1, Sm-D Autoantigen, Small NuclearRibonucleoprotein D1 Polypeptide 16kDa Pseudogene, SnRNP Core ProteinD1, Small Nuclear Ribonucleoprotein Sm D1; SLC35D3 (Solute CarrierFamily 35, Member D3, FRCL1, Fringe Connection-Like Protein 1,bA55K22.3, Frc, Fringe-Like 1, Solute Carrier Family 35 Member D3; NCBIGenBank: NC_000006.11 NC_018917.2 NT_025741.16); SIRT2 (Sirtuin 2,NAD-Dependent Deacetylase Sirtuin-2, SIRL2, Silent Information Regulator2, Regulatory Protein SIR2 Homolog 2, Sir2-Related Protein Type 2,SIR2-Like Protein 2, Sirtuin Type 2, Sirtuin (Silent Mating TypeInformation Regulation 2 Homolog) 2 (S. cerevisiae), Sirtuin-2, Sirtuin(Silent Mating Type Information Regulation 2, S. cerevisiae, Homolog) 2,EC 3.5.1., SIR2; GenBank: AAK51133.1); Sema 5b (FLJ10372, KIAA1445,Mm.42015, SEMASB, SEMAG, Semaphorin 5b Hlog, sema domain, seventhrombospondin repeats (type 1 and type 1-like), transmembrane domain(TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accessionno. AB04087; secernin 1 (SCRN1, SES1, KIAA0193, secerin-1; GenBank:EAL24458.1); SAGE (SAGE1, Sarcoma Antigen 1, Cancer/Testis Antigen 14,CT14, Putative Tumor Antigen; NCBI Reference Sequence: NP_061136.2);RU2AS (KAAG1, Kidney Associated Antigen 1, RU2AS, RU2 Antisense GeneProtein, Kidney-Associated Antigen 1; GenBank: AAF23613.1); RNF43—E3ubiquitin-protein ligase RNF43 precursor [Homo sapiens], RNF124; URCC;NCBI Reference Sequence: NP_060233.3; RhoC (RGSS (Regulator Of G-ProteinSignaling 5, MSTP032, Regulator Of G-Protein Signalling 5, MSTP092,MST092, MSTP106, MST106, MSTP129, MST129; GenBank: AAB84001.1); RET (retproto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114;RET51; RET-ELE; NP_066124.1; NM_020975.4); RBAF600 (UBR4, UbiquitinProtein Ligase E3 Component N-Recognin 4, Zinc Finger, UBR1 Type 1,ZUBR1, E3 Ubiquitin-Protein Ligase UBR4, RBAF600, 600 KDa RetinoblastomaProtein-Associated Factor, Zinc Finger UBR1-Type Protein 1, EC 6.3.2.,N-recognin-4, KIAA0462, p600, EC 6.3.2, KIAA1307; GenBank: AAL83880.1);RAGE-1 (MOK, MOK Protein Kinase, Renal Tumor Antigen, RAGE, MAPK/MAK/MRKOverlapping Kinase, Renal Tumor Antigen 1, Renal Cell Carcinoma Antigen,RAGE-1, EC 2.7.11.22, RAGE1; UniProtKB/Swiss-Prot: Q9UQ07.1);RAB38/NY-MEL-1 (RAB38, NY-MEL-1, RAB38, Member RAS Oncogene Family,Melanoma Antigen NY-MEL-1, Rab-Related GTP-Binding Protein, Ras-RelatedProtein Rab-38, rrGTPbp; GenBank: AAH15808.1); PTPRK (DJ480J14.2.1(Protein Tyrosine Phosphatase, Receptor Type, K R-PTP-KAPPA, ProteinTyrosine Phosphatase Kappa, Protein Tyrosine Phosphatase Kappa), ProteinTyrosine Phosphatase, Receptor Type, K, Protein-Tyrosine PhosphataseKappa, Protein-Tyrosine Phosphatase, Receptor Type, Kappa, R-PTP-kappa,Receptor-Type Tyrosine-Protein Phosphatase Kappa, EC 3.1.3.48, PTPK;GenBank: AAI44514.1); PSMA; PSCA hlg(2700050C12Rik, C530008O16Rik, RIKENcDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.AY358628); PSCA (Prostate stem cell antigen precursor, Genbank accessionno. AJ29743; PRDXS (Peroxiredoxin 5, EC 1.11.1.15, TPx Type VI, B166,Antioxidant Enzyme B166, HEL-S-55, Liver Tissue 2D-Page Spot 71B, PMP20,Peroxisomal Antioxidant Enzyme, PRDX6, Thioredoxin Peroxidase PMP20,PRXV, AOEB166, Epididymis Secretory Protein Li 55, Alu Co-Repressor 1,Peroxiredoxin-5, Mitochondrial, Peroxiredoxin V, prx-V, ThioredoxinReductase, Prx-V, ACR1, Alu Corepressor, PLP; GenBank: CAG33484.1);PRAME (Preferentially Expressed Antigen In Melanoma, PreferentiallyExpressed Antigen Of Melanoma, MAPE, 01P-4, OIPA, CT130, Cancer/TestisAntigen 130, Melanoma Antigen Preferentially Expressed In Tumors,Opa-Interacting Protein 4, Opa-Interacting Protein 01P4; GenBank:CAG30435.1); pml-RARalpha fusion protein; PMEL17 (silver homolog; SILV;D12S53E; PMEL17; SI; SIL); ME20; gp10 BC001414; BT007202; M32295;M77348; NM_006928; PBF (ZNF395, Zinc Finger Protein 395, PRF-1,Huntington disease regulatory, HD Gene Regulatory Region-BindingProtein, Region-Binding Protein 2, Protein 2, Papillomavirus RegulatoryFactor 1, HD-Regulating Factor 2, Papillomavirus-Regulatory Factor,PRF1, HDBP-2, Si-1-8-14, HDBP2, Huntington'S Disease Gene RegulatoryRegion-Binding Protein 2, HDRF-2, Papillomavirus Regulatory FactorPRF-1, PBF; GenBank: AAH01237.1); PAXS (Paired Box 5, Paired BoxHomeotic Gene 5, BSAP, Paired Box Protein Pax-5, B-Cell Lineage SpecificActivator, Paired Domain Gene 5, Paired Box Gene 5 (B-Cell LineageSpecific Activator Protein), B-Cell-Specific Transcription Factor,Paired Box Gene 5 (B-Cell Lineage Specific Activator); PAP (REG3A,Regenerating Islet-Derived 3 Alpha, INGAP, PAP-H, HepatointestinalPancreatic Protein, PBBCGF, Human Proislet Peptide, REG-III,Pancreatitis-Associated Protein 1, Reg3, Reg III-Alpha,hepatocarcinoma-intestine-pancreas, Regenerating Islet-Derived ProteinIII-Alpha, Pancreatic Beta Cell Growth Factor, HIP, PAP HomologousProtein, HIP/PAP, Proliferation-Inducing Protein 34, PAP1,Proliferation-Inducing Protein 42, REG-3-alpha, RegeneratingIslet-Derived Protein 3-Alpha, Pancreatitis-Associated Protein; GenBank:AAH36776.1); p53 (TP53, Tumor Protein P53, TPR53, P53, Cellular TumorAntigen P53, Antigen NY-CO-13, Mutant Tumor Protein 53, PhosphoproteinP53, P53 Tumor Suppressor, BCC7, Transformation-Related Protein 53,LFS1, tumor Protein 53, Li-Fraumeni Syndrome, Tumor Suppressor P53; P2X5(Purinergic receptor P2X ligand-gated ion channel 5, an ion channelgated by extracellular ATP, may be involved in synaptic transmission andneurogenesis, deficiency may contribute to the pathophysiology ofidiopathic detrusor instability); 422 aa), pl: 7.63, MW: 47206 TM: 1 [P]Gene Chromosome: 17p13.3, Genbank accession No. NP_002552.; OGT(0-Linked N-Acetylglucosamine (GlcNAc) Transferase, 0-GlcNAc TransferaseP110 Subunit, 0-Linked N-Acetylglucosamine (GlcNAc) Transferase(UDP—N-Acetylglucosamine:Polypeptide-N-Acetylglucosaminyl Transferase,UDP—N-Acetylglucosamine-Peptide N-Acetylglucosaminyltransferase 110 KDaSubunit, UDP—N-Acetylglucosamine:Polypeptide-N-AcetylglucosaminylTransferase, Uridinediphospho-N-Acetylglucosamine:PolypeptideBeta-N-Acetylglucosaminyl Transferase, 0-GlcNAc Transferase SubunitP110, EC 2.4.1.255, 0-Linked N-Acetylglucosamine Transferase 110 KDaSubunit, EC 2.4.1, HRNT1, EC 2.4.1.186, O-GLCNAC; GenBank: AAH38180.1);0A1 (Osteoarthritis QTL 1, OASD; GenBank: CAA88742.1); NY-ES0-1/LAGE-2(Cancer/Testis Antigen 1B, CTAG1B, NY-ESO-1, LAGE-2, ESO1, CTAG1, CTAG,LAGE2B, Cancer/Testis Antigen 1, Autoimmunogenic Cancer/Testis AntigenNY-ESO-1, Ancer Antigen 3, Cancer/Testis Antigen 6.1, New YorkEsophageal Squamous Cell Carcinoma 1, L Antigen Family Member 2, LAGE2,CT6.1, LAGE2A; GenBank: AA130365.1); NY-BR-1 (ANKRD30A, Ankyrin RepeatDomain 30A, Breast Cancer Antigen NY-BR-1, Serologically Defined BreastCancer Antigen NY-BR-1, Ankyrin Repeat Domain-Containing Protein 30A;NCBI Reference Sequence: NP_443723.2); N-ras (NRAS, Neuroblastoma RASViral (V-Ras) Oncogene Homolog, NRAS1, Transforming Protein N-Ras,GTPase NRas, ALPS4, N-Ras Protein Part 4, NS6, Oncogene Homolog, HRAS1;GenBank: AAH05219.1); NFYC (Nuclear Transcription Factor Y, Gamma, HAPS,HSM, Nuclear Transcription Factor Y Subunit C, Transactivator HSM-1/2,CCAAT Binding Factor Subunit C, NF-YC, CCAAT Transcription BindingFactor Subunit Gamma, CAAT Box DNA-Binding Protein Subunit C, Histone H1Transcription Factor Large Subunit 2A, CBFC, Nuclear TranscriptionFactor Y Subunit Gamma, CBF-C, Transactivator HSM-1, H1TF2A,Transcription Factor NF-Y, C Subunit; neo-PAP (PAPOLG, Poly(A)Polymerase Gamma, Neo-Poly(A) Polymerase, Nuclear Poly(A) PolymeraseGamma, Polynucleotide Adenylyltransferase Gamma, SRP RNA 3′ AdenylatingEnzyme/Pap2, PAP-gamma, Neo-PAP, SRP RNA 3′-Adenylating Enzyme, PAP2, EC2.7.7.19, PAPG; NCBI Reference Sequence: NP_075045.2); NCA (CEACAM6,Genbank accession no. M1872); Napi3b (NAPI-3B, NPTIIb, SLC34A2, solutecarrier family 34 (sodium phosphate), member 2, type II sodium-dependentphosphate transporter 3b, Genbank accession no. NM_00642); Myosin classI; MUM-3; MUM-2 (TRAPPC1, Trafficking Protein Particle Complex 1, BETS,BETS Homolog, MUM2, Melanoma Ubiquitous Mutated 2, Multiple MyelomaProtein 2, Trafficking Protein Particle Complex Subunit 1; MUM-1f; Mucin(MUC1, Mucin 1, Cell Surface Associated, PEMT, PUM, CA 15-3, MCKD1,ADMCKD, Medullary Cystic Kidney Disease 1 (Autosomal Dominant), ADMCKD1,Mucin 1, Transmembrane, CD227, Breast Carcinoma-Associated Antigen DF3,MAM6, Cancer Antigen 15-3, MCD, Carcinoma-Associated Mucin, MCKD, KrebsVon Den Lungen-6, MUC-1/SEC, Peanut-Reactive Urinary Mucin, MUC1/ZD,Tumor-Associated Epithelial Membrane Antigen, DF3 Antigen,Tumor-Associated Mucin, episialin, EMA, H23 Antigen, H23AG, Mucin-1,KL-6, Tumor Associated Epithelial Mucin, MUC-1, Episialin, PEM, CD227Antigen; UniProtKB/Swiss-Prot: P15941.3); MUC5AC (Mucin 5AC, OligomericMucus/Gel-Forming, Tracheobronchial Mucin MUC5, TBM, Mucin 5, Subtypes AAnd C, Tracheobronchial/Gastric, leB, Gastric Mucin, Mucin 5AC,Oligomeric Mucus/Gel-Forming Pseudogene, Lewis B Blood Group Antigen,LeB, Major Airway Glycoprotein, MUC-5AC, Mucin-5 Subtype AC,Tracheobronchial; MUC1 (Mucin 1, Cell Surface Associated, PEMT, PUM, CA15-3, MCKD1, ADMCKD, Medullary Cystic Kidney Disease 1 (AutosomalDominant), ADMCKD1, Mucin 1, Transmembrane, CD227, BreastCarcinoma-Associated Antigen DF3, MAM6, Cancer Antigen 15-3, MCD,Carcinoma-Associated Mucin, MCKD, Krebs Von Den Lungen-6, MUC-1/SEC,Peanut-Reactive Urinary Mucin, MUC-1/X, Polymorphic Epithelial Mucin,MUC1/ZD, Tumor-Associated Epithelial Membrane Antigen, DF3 Antigen,Tumor-Associated Mucin, episialin, EMA, h23 Antigen, H23AG, mucin-1,KL-6, Tumor Associated Epithelial Mucin, MUC-1, Episialin, PEM, CD227Antigen; MSG783 (RNF124, hypothetical protein FLJ20315, Genbankaccession no. NM-01776; MRP4—multidrug resistance-associated protein 4isoform 3, MOAT-B; MOATB [Homo sapiens]; NCBI Reference Sequence:NP_001288758.1; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,mesothelin, Genbank accession no. NM_00582; MMP-7 (MMP7, matrilysin,MPSL1, matrin, Matrix Metalloproteinase 7 (Matrilysin, Uterine), UterineMatrilysin, Matrix Metalloproteinase-7, EC 3.4.24.23, Pump-1 Protease,Matrin, Uterine Metalloproteinase, PUMP1, MMP-7, EC 3.4.24, PUMP-1;GenBank: AAC37543.1); MMP-2 (MMP2, Matrix Metallopeptidase 2 (GelatinaseA, 72kDa Gelatinase, 72kDa Type IV Collagenase), MONA, CLG4A, MatrixMetalloproteinase 2 (Gelatinase A, 72kD Gelatinase, 72kD Type IVCollagenase), CLG4, 72kDa Gelatinase, 72kDa Type IV Collagenase), MatrixMetalloproteinase-2, MMP-II, 72 KDa Gelatinase, Collagenase Type IV-A,MMP-2, Matrix Metalloproteinase-II, TBE-1, Neutrophil Gelatinase, EC3.4.24.24, EC 3.4.24; GenBank: AAH02576.1); and Meloe.

For example, in some instances, an anti-CD3 antibody having a firstbinding domain comprising at least an HVR-L3 sequence of TQSFILRT (SEQID NO: 6) and one, two, three, four, or five HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; and (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 5, may have a secondbinding domain that binds to Ly6G6D. In some instances, the firstbinding domain that binds CD3 comprises at least one (e.g., 1, 2, 3, or4) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4comprising the sequences of SEQ ID NOs: 9-12, respectively, and/or atleast one (e.g., 1, 2, 3, or 4) of the light chain framework regionsFR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs:13-16, respectively. In some instances, the first binding domain thatbinds to CD3 may, for example, comprise a VL domain comprising an aminoacid sequence having at least 90% sequence identity (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 8. In some instances, the first binding domainthat binds to CD3 may, for example, comprise (a) a VL domain comprisingan amino acid sequence having at least 90% sequence identity (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, SEQ ID NO: 8 and (b) a VH domain comprising anamino acid sequence having at least 90% sequence identity (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, SEQ ID NO: 7, such as possessed by the anti-CD3antibody, 38E4v11, described herein.

In other instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 6, such as 38E4v11, mayhave a second binding domain that binds to a cell surface antigen (e.g.,a tumor antigen) on a target cell other than an immune effector cell.

In some instances, the cell surface antigen may be expressed in low copynumber on the target cell. For example, in some instances, the cellsurface antigen is expressed or present at less than 35,000 copies pertarget cell. In some embodiments, the low copy number cell surfaceantigen is present between 100 and 35,000 copies per target cell;between 100 and 30,000 copies per target cell; between 100 and 25,000copies per target cell; between 100 and 20,000 copies per target cell;between 100 and 15,000 copies per target cell; between 100 and 10,000copies per target cell; between 100 and 5,000 copies per target cell;between 100 and 2,000 copies per target cell; between 100 and 1,000copies per target cell; or between 100 and 500 copies per target cell.Copy number of the cell surface antigen can be determined, for example,using a standard Scatchard plot.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 6, such as 38E4v11, mayhave a second binding domain that binds to Ly6G6D. Ly6G6D may beexpressed or present in low copy number on the target cell, for example,at about 20,000 copies per target cell to about 30,000 copies per targetcell (e.g., between about 20,000 copies per target cell to about 25,000copies per target cell, or between about 25,000 copies per target cellto about 30,000 copies per target cell), depending on the target cell.The copy number of Ly6G6D can be determined using a standard Scatchardplot. In some instances, the first binding domain that binds CD3comprises at least one (e.g., 1, 2, 3, or 4) of heavy chain frameworkregions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQID NOs: 9-12, respectively, and/or at least one (e.g., 1, 2, 3, or 4) ofthe light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4comprising the sequences of SEQ ID NOs: 13-16, respectively. In someinstances, the first binding domain that binds to CD3 may, for example,comprise a VL domain comprising an amino acid sequence having at least90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO: 8. Insome instances, the first binding domain that binds to CD3 may, forexample, comprise (a) a VL domain comprising an amino acid sequencehaving at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 8 and (b) a VH domain comprising an amino acid sequencehaving at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 7, such as possessed by the anti-CD3 antibody, 38E4v11,described herein.

In other instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to CD20. The secondbinding domain that binds to CD20 may, for example, comprise at leastone, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 21; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 22, such as possessedby the anti-CD20 antibody, 2H7.v16 (described in U.S. Pat. No.7,799,900). In some instances, the second binding domain that binds CD20comprises at least one (e.g., 1, 2, 3, or 4) of heavy chain frameworkregions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQID NOs: 23-26, respectively, and/or at least one (e.g., 1, 2, 3, or 4)of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4comprising the sequences of SEQ ID NOs: 27-30, respectively. In someinstances, the second binding domain that binds to CD20 may, forexample, comprise (a) a VH domain comprising an amino acid sequencehaving at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 31; (b) a VL domain comprising an amino acid sequencehaving at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 32; or (c) a VH domain as in (a) and a VL domain as in(b), such as possessed by the anti-CD20 antibody, 2H7.v16 (described inU.S. Pat. No. 7,799,900).

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to FcRH5. Thesecond binding domain that binds to FcRH5 may, for example, comprise atleast one, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 37; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 38, such as possessedby the anti-FcRH5 antibody, 1G7. In some instances, the second bindingdomain that binds FcRH5 comprises at least one (e.g., 1, 2, 3, or 4) ofheavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprisingthe sequences of SEQ ID NOs: 39-42, respectively, and/or at least one(e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2,FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 43-46,respectively. In some instances, the second binding domain that binds toFcRH5 may, for example, comprise (a) a VH domain comprising an aminoacid sequence having at least 90% sequence identity (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 47; (b) a VL domain comprising an amino acidsequence having at least 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 48; or (c) a VH domain as in (a) and a VL domainas in (b).

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to PMEL17.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to LY6E.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to CD19.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to CD33.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to CD22.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to CD79A.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to CD79B.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to EDAR.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to GFRA1.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to MRP4.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to RET. The secondbinding domain that binds to RET may, for example, comprise at leastone, two, three, four, five, or six hypervariable regions (HVRs)selected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 49; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 53; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 54, such as possessedby the anti-RET antibody, 41205.v6. In some instances, the secondbinding domain that binds to RET may, for example, comprise (a) a VHdomain comprising an amino acid sequence having at least 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, SEQ ID NO: 55; (b) a VLdomain comprising an amino acid sequence having at least 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, SEQ ID NO: 56; or (c) a VHdomain as in (a) and a VL domain as in (b).

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to Steap1.

In some instances, an anti-CD3 antibody having a first binding domaincomprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, such as38E4v11, may have a second binding domain that binds to TenB2.

In some embodiments, bispecific antibodies may also be used to localizecytotoxic agents to cells which express a tumor antigen, such as a tumorantigen listed in Table 1 (e.g., Ly6G6D, CD20, FcRH5, HER2, LYPD1,PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET,Steap1, or TenB2). Bispecific antibodies can also be prepared asfull-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). “Knob-in-hole” engineering of multispecificantibodies may be utilized to generate a first arm containing a knob anda second arm containing the hole into which the knob of the first armmay bind. The knob of the multispecific antibodies of the invention maybe an anti-CD3 arm in one embodiment. Alternatively, the knob of themultispecific antibodies of the invention may be an anti-target/antigenarm in one embodiment. The hole of the multispecific antibodies of theinvention may be an anti-CD3 arm in one embodiment. Alternatively, thehole of the multispecific antibodies of the invention may be ananti-target/antigen arm in one embodiment. Multispecific antibodies mayalso be engineered using immunoglobulin crossover (also known as Fabdomain exchange or CrossMab format) technology (see eg., WO2009/080253;Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)).Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibodies, or antibody fragments thereof, may also include a “DualActing FAb” or “DAF” comprising an antigen binding site that binds toCD3 as well as another, different antigen (e.g., a second biologicalmolecule) (see, e.g., US 2008/0069820).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the anti-CD3antibodies of the invention (e.g., bispecific anti-CD3 antibodies of theinvention that bind to CD3, e.g., with high affinity (e.g., 38E4v11),and a second biological molecule, e.g., a cell surface antigen, e.g., atumor antigen, such as TDB antibodies of the invention or variantsthereof) are contemplated. For example, it may be desirable to improvethe binding affinity and/or other biological properties of the antibody.Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, for example,antigen-binding.

a. Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 2 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 2 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 2 Exemplary and Preferred Amino Acid Substitutions OriginalExemplary Preferred Residue Substitutions Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val;Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b. Glycosylation Variants

In certain embodiments, anti-CD3 antibodies of the invention (e.g.,bispecific anti-CD3 antibodies of the invention that bind to CD3,preferably with high affinity (e.g., 38E4v11), and a second biologicalmolecule, e.g., a cell surface antigen, e.g., a tumor antigen, such asTDB antibodies of the invention or variants thereof) can be altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to anti-CD3 antibody of theinvention may be conveniently accomplished by altering the amino acidsequence such that one or more glycosylation sites is created orremoved.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, anti-CD3 antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e. g. complex,hybrid and high mannose structures) as measured by MALDI-TOF massspectrometry, as described in WO 2008/077546, for example. Asn297 refersto the asparagine residue located at about position 297 in the Fc region(EU numbering of Fc region residues); however, Asn297 may also belocated about ±3 amino acids upstream or downstream of position 297,i.e., between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Anti-CD3 antibodies variants are further provided with bisectedoligosaccharides, for example, in which a biantennary oligosaccharideattached to the Fc region of the antibody is bisected by GlcNAc. Suchantibody variants may have reduced fucosylation and/or improved ADCCfunction. Examples of such antibody variants are described, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c. Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an anti-CD3 antibody of the invention(e.g., a bispecific anti-CD3 antibody of the invention that binds toCD3, preferably with high affinity (e.g., 38E4v11), and a secondbiological molecule, e.g., a cell surface antigen, e.g., a tumorantigen, such as a TDB antibody of the invention or variant thereof),thereby generating an Fc region variant (see e.g., US 2012/0251531). TheFc region variant may comprise a human Fc region sequence (e.g., a humanIgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acidmodification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an anti-CD3 antibodyvariant that possesses some but not all effector functions, which makeit a desirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays mayalso be carried out to confirm that the antibody is unable to bind Clqand hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al. J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al. Blood.101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie Blood.103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al. Intl. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. Nos. 6,737,056 and 8,219,149). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. Nos. 7,332,581 and 8,219,149).

In certain embodiments, the proline at position 329 of a wild-type humanFc region in the antibody is substituted with glycine or arginine or anamino acid residue large enough to destroy the proline sandwich withinthe Fc/Fc.gamma. receptor interface that is formed between the proline329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcgRIII(Sondermann et al. Nature. 406, 267-273, 2000). In certain embodiments,the antibody comprises at least one further amino acid substitution. Inone embodiment, the further amino acid substitution is S228P, E233P,L234A, L235A, L235E, N297A, N297D, or P331S, and still in anotherembodiment the at least one further amino acid substitution is L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region (see e.g., US 2012/0251531), and still in another embodimentthe at least one further amino acid substitution is L234A and L235A andP329G of the human IgG1 Fc region.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) Clq binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

In some aspects the anti-CD3 antibody (e.g., bispecific anti-CD3antibody) comprises an Fc region comprising an N297G mutation. In someembodiments, the anti-CD3 antibody comprising the N297G mutationcomprises an anti-CD3 arm comprising a first binding domain comprisingthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQID NO: 2; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e)an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and ananti-Ly6G6D arm.

In some embodiments, the anti-CD3 antibody comprising the N297G mutationcomprises an anti-CD3 arm comprising a first binding domain comprising(a) a VH domain comprising an amino acid sequence of SEQ ID NO: 7 and(b) a VL domain comprising an amino acid sequence of SEQ ID NO: 8, andan anti-Ly6G6D arm.

In some embodiments, the anti-CD3 antibody comprising the N297G mutationcomprises one or more heavy chain constant domains, wherein the one ormore heavy chain constant domains are selected from a first CH1 (CH1₁)domain, a first CH2 (CH2₁) domain, a first CH3 (CH3₁) domain, a secondCH1 (CH1₂) domain, second CH2 (CH2₂) domain, and a second CH3 (CH3₂)domain. In some instances, at least one of the one or more heavy chainconstant domains is paired with another heavy chain constant domain. Insome instances, the CH3₁ and CH3₂ domains each comprise a protuberanceor cavity, and wherein the protuberance or cavity in the CH3, domain ispositionable in the cavity or protuberance, respectively, in the CH3₂domain. In some instances, the CH3₁ and CH3₂ domains meet at aninterface between said protuberance and cavity. In some instances, theCH2₁ and CH2₂ domains each comprise a protuberance or cavity, andwherein the protuberance or cavity in the CH2, domain is positionable inthe cavity or protuberance, respectively, in the CH2₂ domain. In otherinstances, the CH2₁ and CH2₂ domains meet at an interface between saidprotuberance and cavity. In some instances, the anti-CD3 antibody is anIgG1 antibody.

In other embodiments, the anti-CD3 antibody comprising the N297Gmutation comprises an anti-CD3 arm comprising a first binding domaincomprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO: 7 and (b) a VL domain comprising an amino acid sequence of SEQ IDNO: 8, and an anti-Ly6G6D arm, wherein (a) the anti-CD3 arm comprisesT3665, L368A, Y407V, and N297G substitution mutations and (b) theanti-Ly6G6D arm comprises T366W and N297G substitution mutations.

d. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, for example, inU.S. Pat. No. 7,521,541.

e. Antibody Derivatives

In certain embodiments, an anti-CD3 antibody of the invention (e.g.,bispecific anti-CD3 antibody of the invention that binds to CD3,preferably with high affinity (e.g., 38E4v11), and a second biologicalmolecule, e.g., a cell surface antigen, e.g., a tumor antigen, such as aTDB antibody of the invention or variant thereof) provided herein may befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

7. Masked Antibody Variants

In certain embodiments, any one of the anti-CD3 antibodies describedherein (e.g., a bispecific anti-CD3 antibody, e.g., a Ly6G6D TDBantibody) may include a polypeptide mask, wherein the polypeptide maskcomprises a masking moiety (MM) comprising the amino acid sequence of atleast amino acid residues 1-3 of SEQ ID NO: 78 (e.g., a polypeptide maskcomprising a MM comprising amino acid residues 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19,1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, or 1-27 of SEQ ID NO: 78), oran N-terminal cyclicized glutamine derivative thereof (e.g., apolypeptide mask comprising a MM comprising5-oxopyrrolidine-2-carboxylic acid (PCA)). For example, in someembodiments, the MM comprises at least the first three amino acidresidues of SEQ ID NO: 78, except that the residue at position 1 is anN-terminal cyclicized glutamine (PCA) residue instead of a glutamineresidue (e.g., the MM comprises the amino acid sequence PCA-D-G). Insome embodiments, the anti-CD3 antibody comprises at least one anti-CD3arm (e.g., an anti-CD3 arm comprising the heavy chain variable (VH)domain and a light chain variable (VL) domain of 38E4v11), and thepolypeptide mask is joined to anti-CD3 arm (e.g., via the VH domain orthe VL domain of the anti-CD3 arm). In some embodiments, the MM isextended at the C-terminus by all or a portion of the remaining sequenceof SEQ ID NO: 78. For example, in some embodiments, the MM comprises theamino acid sequence of at least amino acid residues 1-5 of SEQ ID NO:78, or an N-terminal cyclicized glutamine derivative thereof (e.g., apolypeptide mask comprising a MM comprising at least the first fiveamino acid residues of SEQ ID NO: 1, except that the residue at position1 is PCA instead of a glutamine). In other embodiments, the MM comprisesthe amino acid sequence of at least amino acid residues 1-6 of SEQ IDNO: 78, or an N-terminal cyclicized glutamine derivative thereof (e.g.,a polypeptide mask comprising a MM comprising at least the first sixamino acid residues of SEQ ID NO: 78, except that the residue atposition 1 is PCA instead of glutamine). In some embodiments, theanti-CD3 antibody and MM are positioned relative to each other in anN-terminal to C-terminal direction as (MM)-(anti-CD3 antibody). In someinstances, the MM is extended, either directly or indirectly, at one end(i.e., the C-terminal end) by a non-native CD3 polypeptide sequence,such as a cleavable moiety (CM) and/or linker moiety (LM).

As noted above, the masked anti-CD3 antibody (e.g., a masked bispecificanti-CD3 antibody, e.g., a masked Ly6G6D TDB antibody) may comprise apolypeptide mask having both a MM and a cleavable moiety (CM). In someembodiments, the CM contains an amino acid sequence that is capable ofbeing cleaved by an enzyme, such as a protease. In other embodiments,the CM provides a cysteine-cysteine disulfide bond that is cleavable byreduction. In additional embodiments, the CM provides an acid-labilelinker that is cleaved in the presence of an acidic pH environment. Inyet other embodiments, the CM provides a photolytic substrate that isactivatable by photolysis.

Accordingly, a masked anti-CD3 antibody (e.g., a masked Ly6G6D TDBantibody) comprising a polypeptide mask with a CM can exist in either acleaved state or an uncleaved state. As used herein, the term cleavedstate refers to the condition of the anti-CD3 antibody followingmodification of the CM, for example, by a protease, reduction of acysteine-cysteine disulfide bond of the CM, and/or photoactivation. Theterm uncleaved state, as used herein, refers to the condition of theanti-CD3 antibody in the absence of cleavage of the CM, for example, bya protease, in the absence reduction of a cysteine-cysteine disulfidebond of the CM, in the absence of an acidic pH environment (e.g., in aneutral or basic pH environment), and/or in the absence of light. Itwill be apparent to the ordinarily skilled artisan that in someembodiments a cleaved anti-CD3 antibody may lack an MM due to cleavageof the CM by, for example, a protease, resulting in release of at leastthe MM. Thus, when a masked anti-CD3 antibody is in the uncleaved state,the masked anti-CD3 antibody would show reduced binding to CD3 becausethe binding domain of the antibody is effectively masked from the CD3target molecule. In the cleaved state, the anti-CD3 antibody would showhigher affinity for CD3 than an antibody it would in its uncleaved statebecause the binding domain of the antibody would no longer be inhibitedby the MM of the polypeptide mask.

When the CM is capable of being cleaved by an enzyme (e.g., a protease)and the masked anti-CD3 antibody is a TDB, the enzyme may be selectedbased on a protease that is co-localized in tissue with the desiredtarget of the TDB. A variety of different conditions are known in whicha target of interest is co-localized with a protease, where thesubstrate of the protease is known in the art. In the example of cancer,the target tissue can be a cancerous tissue, particularly canceroustissue of a solid tumor. Increased levels of proteases having knownsubstrates in a number of cancers, such as solid tumors, are known inthe art (see, e.g., La Rocca et al. British J. of Cancer. 90(7):1414-1421, 2004 and Lopez-Otin et al. Nat Rev Cancer. 7: 800-808, 2007).Exemplary CMs can include, but are not limited to, substrates that arecleavable by one or more of the enzymes (e.g., proteases) specified inWO 2010/081173, WO 2009/025846, WO 2010/096838, and/or one or more ofthe following enzymes listed below in Table 3.

TABLE 3 Exemplary Enzymes Legumain asparaginyl Transmembrane Plasminendopeptidase Protease Serine (TMPRSS-3/4) Matrix Metalloprotease MMP-2MMP-3 MMP-7 (MMP)-1 MMP-8 MMP-9 MMP-12 MMP-13 MMP-14 Membrane type 1matrix Cathepsin A Cathepsin B metalloprotease (MT1- MMP) Cathepsin DCathepsin E Cathepsin F Cathepsin H Cathepsin K Cathepsin K Cathepsin LCathepsin L2 Cathepsin O Cathepsin S Caspase 1 Caspase 2 Caspase 3Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10Caspase 11 Caspase 12 Caspase 13 Caspase 14 Human NeutrophilUrokinase/urokinase- A Disintegrin and ADAM12 Elastase Type PlasminogenMetalloprotease Activator (uPA) (ADAM)10 ADAM17 ADAM with ADAMTS5 BetaSecretase (BACE) Thrombospondin Motifs (ADAMTS) Fibroblast ActivationGranzyme A Granzyme B Guanidinobenzoatase Protease (FAP) GepsinMatriptase Matriptase 2 Meprin Neprilysin Prostate-Specific TumorNecrosis Factor- Kallikrein-Related Membrane Antigen Converting EnzymePeptidase (KLK)3 (PSMA) (TACE) KLK5 KLK7 KLK11 NS3/4 Protease ofHepatitis C Virus (HCV- NS3/4) Tissue Plasminogen Calpain Calpain 2Glutamate Activator (tPA) Carboxypeptidase II Plasma KallikreinAMSH-Like Protease AMSH γ-Secretase Component Antiplasmin CleavingDecysin 1 Apoptosis-Related N-Acetylated Alpha- Enzyme (APCE) CysteinePeptidase Linked Acidic Dipeptidase-Like 1 Thrombin

Alternatively or in addition, the masked anti-CD3 antibody can comprisea CM that includes a disulfide bond of a cysteine pair, which is thuscleavable by a reducing agent. These include, but are not limited to, acellular reducing agent such as glutathione (GSH), thioredoxins, NADPH,flavins, ascorbate, and the like, which can be present in large amountsin tissue of or surrounding a solid tumor.

In other embodiments, the masked anti-CD3 antibody can comprise a CMthat includes an acid-labile linker (e.g., a hydrazone, an imino, anester, or an amido group) which is thus cleavable in the presence of anacidic pH environment, as described in PCT publication number WO2006/108052, which is herein incorporated by reference in its entirety.This includes, but is not limited to, an acidic pH environment that canbe present in lysosomes of a cell or in a tumor microenvironment.

The CM may be positioned relative to the anti-CD3 antibody and MM in anN-terminal to C-terminal direction as (MM)-(CM)-(anti-CD3 antibody).

In other embodiments, the masked anti-CD3 antibody (e.g., a maskedLy6G6D TDB antibody) can include one or more (e.g., 2 or 3 or more)distinct CMs within its polypeptide mask.

As noted above, the masked anti-CD3 antibody (e.g., a masked Ly6G6D TDBantibody) may comprise a polypeptide mask having both a MM and a linkermoiety (LM), or, alternatively, all three moieties (i.e., a MM, LM, andCM). LMs suitable for use in a polypeptide mask described herein aregenerally ones that provide flexibility and/or length to the mask tofacilitate or modulate the degree of inhibition of the binding of theanti-CD3 antibody to CD3. Such LMs can also be referred to as flexiblelinkers. Suitable LMs can be readily selected and can be of differentsuitable lengths, such as from 1 amino acid (e.g., one glycine (G) orone serine (S) residue) to 30 amino acids (e.g., a LM containing a GSrepeat sequence). A LM is preferably greater than one amino acid inlength (e.g, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more amino acids inlength). In some instances, the LM can be between 5 to 24 amino acids inlength, such as between 5 to 15 amino acids in length. The LM may highin G and/or S content (i.e. a G/S-rich LM) and may include GS repeats.For example, the LM may include glycine polymers (G)n, glycine-serinepolymers (including, for example, (GS)n, (GSGGS)n and (GGGS)n, where nis an integer of at least one), glycine-alanine polymers, alanine-serinepolymers, and other flexible linker combinations known in the art.Glycine and glycine-serine polymers are relatively unstructured, andtherefore may be able to serve as a neutral LM that indirectly ordirectly joins the MM component of the polypeptide mask to the anti-CD3antibody. Glycine accesses significantly more phi-psi space than evenalanine, and is much less restricted than residues with longer sidechains (see, e.g., Scheraga. Rev. Computational Chem. 11173-142, 1992).Exemplary flexible linkers include, but are not limited toGly-Gly-Ser-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Gly-Ser-Gly,Gly-Ser-Gly-Gly-Gly, Gly-Gly-Gly-Ser-Gly, Gly-Ser-Ser-Ser-Gly, and thelike. The ordinarily skilled artisan will recognize that design of apolypeptide mask can include a LM that is completely or partiallyflexible. For example, a LM may include a flexible portion as well asone or more portions that confer less flexible structure to yield amasked anti-CD3 antibody exhibiting a desired degree of inhibition ofCD3 binding, which can be assessed using, for example, an assay such asthe phage binding ELISA described in detail below.

When the polypeptide mask does not include a CM, the LM may bepositioned relative to the anti-CD3 antibody and MM in an N-terminal toC-terminal direction as (MM)-(LM)-(anti-CD3 antibody). When thepolypeptide mask does include a CM, the LM may be positioned relative tothe anti-CD3 antibody, MM, and CM in an N-terminal to C-terminaldirection as (MM)-(LM)-(CM)-(anti-CD3 antibody) or(MM)-(CM)-(LM)-(anti-CD3 antibody).

In other embodiments, the masked anti-CD3 antibody (e.g., a maskedLy6G6D TDB antibody) can include one or more (e.g., 2 or 3 or more)distinct CMs within its polypeptide mask.

B. Recombinant Methods and Compositions

Anti-CD3 antibodies of the invention (e.g., bispecific anti-CD3antibodies of the invention that bind to CD3, preferably with highaffinity (e.g., 38E4v11), and a second biological molecule, e.g., a cellsurface antigen, e.g., a tumor antigen, such as TDB antibodies of theinvention or variants thereof) may be produced using recombinant methodsand compositions, for example, as described in U.S. Pat. No. 4,816,567.In one embodiment, isolated nucleic acid encoding an anti-CD3 antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antibody (e.g., the light and/or heavy chains of theantibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In oneembodiment, a method of making an anti-CD3 antibody is provided, whereinthe method comprises culturing a host cell comprising a nucleic acidencoding the antibody, as provided above, under conditions suitable forexpression of the antibody, and optionally recovering the antibody fromthe host cell (or host cell culture medium).

For recombinant production of an anti-CD3 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells.

Numerous baculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas YO, NSO and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-CD3 antibodies of the invention (e.g., bispecific anti-CD3antibodies of the invention that bind to CD3, preferably with highaffinity (e.g., 38E4v11), and a second biological molecule, e.g., a cellsurface antigen, e.g., a tumor antigen, such as TDB antibodies of theinvention or variants thereof) provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an anti-CD3 antibody of the invention is tested for itsantigen binding activity, for example, by known methods such as ELISA,Western blot, etc.

In another aspect, competition assays may be used to identify anantibody that competes with an anti-CD3 antibody of the invention forbinding to CD3.

In an exemplary competition assay, immobilized CD3 is incubated in asolution comprising a first labeled antibody that binds to CD3 and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to CD3. The second antibodymay be present in a hybridoma supernatant. As a control, immobilized CD3is incubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to CD3, excess unboundantibody is removed, and the amount of label associated with immobilizedCD3 is measured. If the amount of label associated with immobilized CD3is substantially reduced in the test sample relative to the controlsample, then that indicates that the second antibody is competing withthe first antibody for binding to CD3. See, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual. Ch.14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-CD3 antibodiesthereof having biological activity. Biological activity may include, forexample, binding to CD3 (e.g., CD3 on the surface of a T cell), or apeptide fragment thereof, either in vivo, in vitro, or ex vivo. In thecase of a multispecific (e.g., bispecific) anti-CD3 antibody of theinvention (e.g., a TDB antibody having one anti-CD3 arm, e.g., 38E4v11,and one arm that recognizes a second biological molecule, e.g., a cellsurface antigen, e.g., a tumor antigen), biological activity may alsoinclude, for example, effector cell activation (e.g., T cell (e.g., CD8+and/or CD4+ T cell) activation), effector cell population expansion(i.e., an increase in T cell count), target cell population reduction(i.e., a decrease in the population of cells expressing the secondbiological molecule on their cell surfaces), and/or target cell killing.Antibodies having such biological activity in vivo and/or in vitro areprovided. In certain embodiments, an antibody of the invention is testedfor such biological activity, as described in detail in the Example 2herein below.

Further, cells may be washed in RPMI medium containing 10% FBS,supplemented with GlutaMax, penicillin & streptomycin, and ˜0.2 millionsuspended cells added to a 96-well U-bottom plate. Cells may be culturedin RPMI1640 supplemented with 10% FBS at 37° C. in a humidified standardcell culture incubator. For BJAB cell killing assays, 20,000 BJAB cellsmay be incubated with effector cells, either as huPBMCs or purified Tcells, as indicated ratios per assay, in the presence of variousconcentrations of TDB antibodies for 24 hours. For endogenous B cellkilling assays, 200,000 huPBMCs may be incubated with variousconcentrations of TDB antibodies for 24 hours.

After culturing, cells may be washed with FACS buffer (0.5% BSA, 0.05%Na Azide in PBS). Cells may then be stained in FACS buffer, washed withFACS buffer and suspended in 100 μl of FACS buffer containing 1 μg/mlPropidium Iodide. Data may be collected on a FACSCalibur flow cytometerand analyzed using FlowJo. Live B cells may be gated out as PI-CD19+ orPI-CD20+ B cells by FACS, and absolute cell count may be obtained withFITC beads added to reaction mix as an internal counting control. Thepercent (%) of cell killing may be calculated based on non-TDB treatedcontrols. Activated T cells may be detected by CD69 and CD25 surfaceexpression using anti-CD69-FITC and anti-CD25-PE.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD3antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an anti-CD3 antibodyas described herein conjugated to an enzymatically active toxin orfragment thereof, including but not limited to diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an anti-CD3 antibodyas described herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-CD3 antibodies of the invention(e.g., bispecific anti-CD3 antibodies of the invention that bind to CD3,preferably with high affinity (e.g., 38E4v11), and a second biologicalmolecule, e.g., a cell surface antigen, e.g., a tumor antigen, such asTDB antibodies of the invention or variants thereof) is useful fordetecting the presence of CD3 in a biological sample. The term“detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue.

In one embodiment, an anti-CD3 antibody for use in a method of diagnosisor detection is provided. In a further aspect, a method of detecting thepresence of CD3 in a biological sample is provided. In certainembodiments, the method comprises contacting the biological sample withan anti-CD3 antibody as described herein under conditions permissive forbinding of the anti-CD3 antibody to CD3, and detecting whether a complexis formed between the anti-CD3 antibody and CD3. Such method may be anin vitro or in vivo method.

In certain embodiments, labeled anti-CD3 antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴O, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-CD3 antibody of the invention(e.g., bispecific anti-CD3 antibody of the invention that binds to CD3,preferably with high affinity (e.g., 38E4v11), and a second biologicalmolecule, e.g., a cell surface antigen, e.g., a tumor antigen, such as aTDB antibody of the invention or variant thereof) are prepared by mixingsuch antibody having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide an additionaltherapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, agrowth inhibitory agent, and/or an anti-hormonal agent, such as thoserecited herein above). Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, for example, films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-CD3 antibodies of the invention (e.g., bispecificanti-CD3 antibodies of the invention that bind to CD3, preferably withhigh affinity (e.g., 38E4v11), and a second biological molecule, e.g., acell surface antigen, e.g., a tumor antigen, may be used in therapeuticmethods. The high affinity anti-CD3 antibodies of the inventionaffinity, for example, 38E4v11, find particular use when paired with atargeting arm that binds to a cell surface antigen that is expressed atlow copy number on tumor cells. In some embodiments, the low copy numbercell surface antigen is Ly6G6D.

In one aspect, an anti-CD3 antibody for use as a medicament is provided.In further aspects, an anti-CD3 antibody for use in treating or delayingprogression of a cell proliferative disorder (e.g., cancer, e.g.,esophageal cancer or an adenocarcinoma) or an autoimmune disorder (e.g.,arthritis) is provided. In certain embodiments, an anti-CD3 antibody foruse in a method of treatment is provided. In certain embodiments, theinvention provides an anti-CD3 antibody for use in a method of treatingan individual having a cell proliferative disorder or an autoimmunedisorder comprising administering to the individual an effective amountof the anti-CD3 antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, for example, as described below.In further embodiments, the invention provides an anti-CD3 antibody foruse in enhancing immune function in an individual having a cellproliferative disorder or an autoimmune disorder. In certainembodiments, the invention provides an anti-CD3 antibody for use in amethod of enhancing immune function in an individual having a cellproliferative disorder or an autoimmune disorder comprisingadministering to the individual an effective of the anti-CD3 antibody toactivate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells),expand (increase) an effector cell population, reduce a target cell(e.g., a cell expressing a second biological molecule recognized by ananti-CD3 antibody of the invention, such as a bispecific TDB antibody ofthe invention) population, and/or kill a target cell (e.g., target tumorcell). An “individual” according to any of the above embodiments may bea human.

In a further aspect, the invention provides for the use of an anti-CD3antibody in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of a cell proliferativedisorder (e.g., cancer, e.g., esophageal cancer or an adenocarcinoma) oran autoimmune disorder (e.g., arthritis). In a further embodiment, themedicament is for use in a method of treating a cell proliferativedisorder or an autoimmune disorder comprising administering to anindividual having a cell proliferative disorder or an autoimmunedisorder an effective amount of the medicament. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, forexample, as described below. In a further embodiment, the medicament isfor activating effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ Tcells), expanding (increasing) an effector cell population, reducing atarget cell (e.g., a cell expressing a second biological moleculerecognized by an anti-CD3 antibody of the invention, such as abispecific TDB antibody of the invention) population, and/or killingtarget cells (e.g., target tumor cells) in the individual. In a furtherembodiment, the medicament is for use in a method of enhancing immunefunction in an individual having a cell proliferative disorder or anautoimmune disorder comprising administering to the individual an amounteffective of the medicament to activate effector cells (e.g., T cells,e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cellpopulation, reduce a target cell (e.g., a cell expressing a secondbiological molecule recognized by an anti-CD3 antibody of the invention,such as a bispecific TDB antibody of the invention) population, and/orkill a target cell (e.g., target tumor cell). An “individual” accordingto any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating a cellproliferative disorder (e.g., cancer, e.g., esophageal cancer or anadenocarcinoma) or an autoimmune disorder (e.g., arthritis). In oneembodiment, the method comprises administering to an individual havingsuch a cell proliferative disorder or an autoimmune disorder aneffective amount of an anti-CD3 antibody. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, for example, asdescribed below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for enhancingimmune function in an individual having a cell proliferative disorder oran autoimmune disorder in an individual having a cell proliferativedisorder or an autoimmune disorder. In one embodiment, the methodcomprises administering to the individual an effective amount of ananti-CD3 antibody to activate effector cells (e.g., T cells, e.g., CD8+and/or CD4+ T cells), expand (increase) an effector cell population,reduce a target cell (e.g., a cell expressing a second biologicalmolecule recognized by an anti-CD3 antibody of the invention, such as abispecific TDB antibody of the invention) population, and/or kill atarget cell (e.g., target tumor cell). In one embodiment, an“individual” is a human.

In a further aspect, the invention provides a method for treatingesophageal cancer, stomach cancer, small intestine cancer, largeintestine cancer, colorectal cancer, or an adenocarcinoma (e.g.,colorectal adenocarcinoma, gastric adenocarcinoma, or pancreaticadenocarcinoma), which may be metastatic adenocarcinoma (e.g.,metastatic colorectal adenocarcinoma, metastatic gastric adenocarcinoma,or metastatic pancreatic adenocarcinoma), by administering an effectiveamount of an anti-CD3 antibody of the invention, such as a bispecificTDB antibody of the invention, such as an anti-Ly6G6D targeting TDB,such as a Ly6G6D TDB having an affinity matured (e.g., high affinity)anti-CD3 arm, such as 38E4v11, and an anti-Ly6G6D arm. In otherembodiments, a Ly6G6D TDB is co-administered (concurrently, as a singleor multiple compositions (e.g., formulations)) with one or moreadditional therapeutic agents, such as any one, two, three, four, five,six, seven, eight, nine, ten, or all eleven of the following: FOLFOX(oxaliplatin (ELOXATIN™) combined with 5-fluorouracil and leucovorin),capecitabine (XELODA®), 5-fluorouracil (5-FU), CapeOx (XELOX;capecitabine with oxaliplatin), leucovorin (folinic acid), bevacizumab(AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), regorafenib(STIVARGA®), irinotecan (CPT-11; CAMPTOSAR®), and FLOX (5-fluorouracilwith oxaliplatin). In other embodiments, a Ly6G6D TDB is administeredbefore one or more additional therapeutic agents, such as any one, two,three, four, five, six, seven, eight, nine, ten, or all eleven of thefollowing: FOLFOX (oxaliplatin (ELOXATIN™) combined with 5-fluorouraciland leucovorin), capecitabine (XELODA®), 5-fluorouracil (5-FU), CapeOx(XELOX; capecitabine with oxaliplatin), leucovorin (folinic acid),bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®),regorafenib (STIVARGA®), irinotecan (CPT-11; CAMPTOSAR®), and FLOX(5-fluorouracil with oxaliplatin). In other embodiments, a Ly6G6D TDB isadministered after one or more additional therapeutic agents, such asany one, two, three, four, five, six, seven, eight, nine, ten, or alleleven of the following: FOLFOX (oxaliplatin (ELOXATIN™) combined with5-fluorouracil and leucovorin), capecitabine (XELODA®), 5-fluorouracil(5-FU), CapeOx (XELOX; capecitabine with oxaliplatin), leucovorin(folinic acid), bevacizumab (AVASTIN®), cetuximab (ERBITUX®),panitumumab (VECTIBIX®), regorafenib (STIVARGA®), irinotecan (CPT-11;CAMPTOSAR®), and FLOX (5-fluorouracil with oxaliplatin).

In another aspect, the invention provides a method for treating ahematological cancer, such as a B cell cancer (for example, matureB-cell lymphoma) by administering an effective amount of an anti-CD3antibody of the invention, such as a bispecific TDB antibody of theinvention, such as an anti-B cell targeting TDB, such as a CD20-TDBhaving an anti-CD3 arm and an anti-CD20 arm. In a further aspect of theembodiment, the mature B-cell lymphoma is a Non-Hodgkin's Lymphoma(NHL). In a further aspect of the embodiment, the NHL is selected fromthe group comprising: germinal-center B-cell-like (GCB) DLBCL, activatedB-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma(MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL),marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL),lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM),central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cellprolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cellleukemia, Splenic lymphoma/leukemia, unclassifiable, Splenic diffuse redpulp small B-cell lymphoma, Hairy cell leukemia variant, WaldenstrOmmacroglobulinemia, Heavy chain diseases, a Heavy chain disease, γ Heavychain disease, μ Heavy chain disease, Plasma cell myeloma, Solitaryplasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginalzone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma),Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma,Pediatric follicular lymphoma, Primary cutaneous follicle centrelymphoma, T-cell/histiocyte rich large B-cell lymphoma, Primary DLBCL ofthe CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of theelderly, DLBCL associated with chronic inflammation, Lymphomatoidgranulomatosis, Primary mediastinal (thymic) large B-cell lymphoma,Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma,Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, Primary effusion lymphoma: B-celllymphoma, unclassifiable, with features intermediate between diffuselarge B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma,unclassifiable, with features intermediate between diffuse large B-celllymphoma and classical Hodgkin lymphoma. In a preferred embodiment ofthe invention, the method comprises treating a cancer comprisinggerminal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC)DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acutemyeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zonelymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacyticlymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervoussystem lymphoma (CNSL), or Burkitt's lymphoma (BL).

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-CD3 antibodies provided herein, e.g., for usein any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-CD3 antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-CD3antibodies provided herein and at least one additional therapeuticagent, for example, as described herein.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a chemotherapeutic agent, growth inhibitory agent, cytotoxicagent, agent used in radiation therapy, anti-angiogenesis agent,apoptotic agent, anti-tubulin agent, or other agent, such as a epidermalgrowth factor receptor (EGFR) antagonist (e.g., a tyrosine kinaseinhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva™), plateletderived growth factor inhibitor (e.g., Gleevec™ (Imatinib Mesylate)), aCOX-2 inhibitor (e.g., celecoxib), interferon, cytokine, antibody otherthan the anti-CD3 antibody of the invention, such as an antibody thatbind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA VEGF, or VEGF receptor(s), TRAIL/Apo2, PD-1,PD-L1, PD-L2, or another bioactive or organic chemical agent.

In some embodiments, the invention provides a method wherein theadditional therapeutic agent is a glucocorticoid. In one embodiment, theglucocorticoid is dexamethasone.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-CD3 antibody and administration of an additional therapeutic agentoccur within about one month, or within about one, two or three weeks,or within about one, two, three, four, five, or six days, of each other.Anti-CD3 antibodies of the invention (e.g., bispecific anti-CD3antibodies of the invention that bind to CD3 and a second biologicalmolecule, e.g., a cell surface antigen, e.g., a tumor antigen, such as aTDB antibody of the invention or variant thereof) can also be used incombination with radiation therapy.

An antibody of the invention (and/or any additional therapeutic agent)can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, the antibody isadministered by subcutaneous administration. In some embodiments, ananti-CD3 antibody administered by subcutaneous injection exhibits a lesstoxic response in a patient than the same anti-CD3 antibody administedby intravenous injection. Dosing can be by any suitable route, forexample, by injections, such as intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments.

As a general proposition, the therapeutically effective amount of theanti-CD3 antibody administered to human will be in the range of about0.01 to about 100 mg/kg of patient body weight whether by one or moreadministrations. In some embodiments, the antibody used is about 0.01 toabout 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 toabout 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1mg/kg administered daily, for example. In one embodiment, an anti-CD3antibody described herein is administered to a human at a dose of about100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of21-day cycles. The dose may be administered as a single dose or asmultiple doses (e.g., 2 or 3 doses), such as infusions. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody wouldbe in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one ormore doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, for example, every week or every threeweeks (e.g., such that the patient receives from about two to abouttwenty, or, for example, about six doses of the anti-CD3 antibody). Aninitial higher loading dose, followed by one or more lower doses may beadministered. The progress of this therapy is easily monitored byconventional techniques and assays.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy may be aseparate administration of one or more of the thereapeutic agentsdescribed above.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Generation of a Affinity Matured Anti-CD3 Antibody NNKLibrary Construction and Panning

To further improve the affinity of the humanized anti-CD3 antibody,38E4v1 (having HVR-H1 to -H3 and HVR-L1 to -L3 of SEQ ID NOs: 57-62,respectively, and a VH and VL sequence of SEQ ID NOs: 63 and 64), phagelibraries were constructed from variant 38E4v1 in Fab-amber format formonovalent Fab phage display with either light chain HVR residues (i.e.,HVR-L1, HVR-L2, and HVR-L3) or heavy chain HVR residues (i.e., HVR-H1,HVR-H2, and HVR-H3) randomized using the NNKdegenerate codon, whichencodes for all 20 amino acids with 32 codons (see, e.g., Brenner et al.Proc. Natl. Acad. Sci. USA 89(12): 5381-5383, 1992). Libraries weredesigned to allow one NNK mutation in each of the three light chain orheavy chain HVRs. The resultant library DNA was electroporated into E.coli XL1 cells, yielding approximately 10⁹ transformants.

Phage libraries were incubated in PBS with 1% BSA and 0.05% Tween for 30minutes and then applied on a CD3ε immobilized plate for first roundpanning. In the subsequent two rounds, decreased concentrations ofbiotinylated CD3ε antigen were used with 1000x non-biotinylated CD3ε ascompetitor in superblock buffer to increase the selection stringency.The phagemids of the third round panning were prepped for deepsequencing analysis.

Deep Sequencing of 38E4v1 Affinity Maturation Libraries

For deep sequencing, phagemid double-stranded DNA was isolated from E.coli XL-1 cells carrying phagemid vectors from the initial phage libraryand the third round of selection. Purified DNA was used as template fora limited cycle PCR-based amplification of VL and VH regions usingPHUSION® DNA polymerase (New England Biolabs). PCR products werepurified by agarose gel extraction and clean-up (Qiagen Gel ExtractionKit). Eluted amplicon DNA was used as the basis for deep sequencinglibrary preparation with standard Illumina library preparation methods,using a TRUSEQ™ DNA Sample Prep kit (Illumina). Adapter-ligatedlibraries were subjected to a single cycle of PCR and sequenced on theIllumina MISEQ®, using paired-end sequencing with an insert size of 200bp or 300 bp, as appropriate, to cover the entire length of theamplicon.

Deep Sequencing Analysis of 38E4v1 Affinity Maturation Libraries

Sequencing data were analyzed using the statistical programming languageR (see, e.g., R Core Team, R: A language and environment for statisticalcomputing, 2013) and the ShortRead package (see Morgan et al.Bioinformatics 25(19): 2607-2608). Quality control was performed onidentified HVR sequences. Each HVR sequence was checked for the correctlength and was allowed to carry only up to one NNK mutation and nonon-NNK mutation. Position weight matrices were generated by calculatingthe frequency of all mutations of every randomized position. Enrichmentratios for each mutation were calculated by dividing the frequency of agiven mutation at a given position in the sorted sample with thefrequency of the very same mutation in the unsorted sample, as describedpreviously (Fowler et al. Nature Methods 7(9): 741-746, 2010). Thesingle mutation from the light chain and heavy chain libraries with highenrichment ratio were selected to synthesize to clone into an mammalianFab expression construct with Flag tag. The plasmids with both heavy andlight chain were transfected into 293T cells for 30 ml expression andFabs were purified using an anti-Flag column.

Fab Affinity Determination by BIACORE® SPR

The binding affinity of selected Fab variants for CD3ε was measured bySPR with a BIACORE® T200 instrument. Briefly, a series S sensor chip CM5was activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)and N-hydroxysuccinimide (NHS) reagents according to the supplier'sinstructions, and 5 μg/ml CD3ε in 10 mM sodium acetate (pH4.0) wascoupled to achieve 100 response units (RU), except for FC1 (which servedas a reference). Unreacted groups were then blocked with 1 Methanolamine. Next, three-fold serial dilutions of Fab in HBS-P buffer(0.01 M HEPES pH 7.4, 0.15M NaCl, 0.005% surfactant P20) from low (0.02nM) to high (20 nM) were injected (flow rate: 30 μl/min). The bindingresponses on Fab were corrected by subtracting of RU from a blank flowcell. The sensorgram was recorded and subject to reference and buffersubtraction before evaluating by BIACORE® T200 Evaluation Software(Version 2.0). Association rates (k_(a)) and dissociation rates (k_(d))were calculated using a simple one-to-one Langmuir binding model. Theequilibrium dissociation constant (K_(D)) was calculated as the ratio ofk_(d)/k_(a). The binding affinity kinetics were determined for ahigh-affinity anti-CD3 Fab, 38E4v11, having the heavy chain and lightchain variable domain sequences set forth in FIG. 1. Compared to that ofits parent, 38E4v11 displayed more than a 10-fold increase in bindingaffinity for CD3 (i.e., a greater than 10-fold decrease in K_(D)compared to 38E4v1) (Table 4).

TABLE 4 38E4v11 binding affinity kinetics relative to 38E4v1 Samplek_(a) (1/M · s) k_(d) (1/s) K_(D) (nM) 38E4v11   5 × 10⁷  1.6 × 10⁻³0.032 38E4v1 (parental Fab) 8.15 × 10⁶ 3.17 × 10⁻³ 0.389

Example 2. Generation and Characterization of Exemplary Ly6G6D/CD3 TDBs(Ly6G6D TDBs)

Given the high affinity of 38E4v11 towards CD3, we tested its utility inthe context of a T cell-targeting therapeutic antibody, also referred toas a “T cell-dependent bispecific” (TDB) antibody. TDB antibodies arecapable of simultaneously binding cell surface antigens on T cells(e.g., CD3) and cell surface antigens on tumor cells, thereby enablingthe bound T cells to contribute to the destruction of the tumor cells.However, TDB antibodies directed to a low copy number cell surfaceantigen target on the surface of tumor cells may exhibit decreasedefficacy. Therefore, TDB antibodies having a high-affinity arm directedto a cell surface antigen on T cells, such as CD3, may be particularlyuseful for pairing with an arm directed to a low copy number target.

To test the utility of the high-affinity 38E4v11 in such a context, wegenerated a Ly6G6D TDB antibody having one high-affinity arm directed toCD3 (38E4v11) and one arm directed to the cell surface antigen Ly6G6D,which is often expressed in low copy number or inconsistently on thesurface of certain tumors (see, e.g., U.S. Pat. No. 7,951,546, which isherein incorporated by reference in its entirety). The Ly6G6D TDBs wereproduced as full-length antibodies in the knob-into-hole format as humanIgG1 (Atwell et al. J. Mol. Biol. 270: 26-35, 1997). Half antibodieswere expressed in either E. coli or Chinese hamster ovary (CHO) cells,purified by Protein A-affinity chromatography, and the proper halfantibody pairs were annealed in vitro as described previously (Spiess etal. Nat. Biotechnol. 2013). If TDB antibody production was carried outin CHO cells, the antibody included an aglycosylation mutation, forexample, at residue N297 (e.g., N297G), such that the TDB antibody wasan effector-less variant and unable to initiate antibody-dependentcell-mediated cytotoxicity (ADCC). After annealing, the Ly6G6D TDBs werepurified by Hydrophobic Interaction Chromatography (HIC) andcharacterized by analytical gel filtration, mass spectrometry, andpolyacrylamide gel electrophoresis. By these methods, a Ly6G6D TDBhaving 38E4v11 as its anti-CD3 arm was generated, which is referred toherein as Ly6G6D (38E4v11) TDB. In addition, two other Ly6G6D TDBs,referred to herein as Ly6G6D (40G5c) TDB and Ly6G6D (38E4v1) TDB, whichshare the same anti-Ly6G6D arm but possess either an anti-CD3 arm 40G5c(having HVR-H1 to -H3 and HVR-L1 to -L3 of SEQ ID NOs: 65-70,respectively, and a VH and VL sequence of SEQ ID NOs: 71 and 72) or ananti-CD3 arm 38E4v1 (described above), respectively, were alsogenerated.

The three Ly6G6D TDBs (Ly6G6D (38E4v11) TDB, Ly6G6D (40G5c) TDB, andLy6G6D (38E4v1) TDB) were then tested in in vitro cytotoxicity assaysand T cell activation assays. For cell killing assays, 15,000 293 cellsoverexpressing human Ly6G6D antigens (293-Ly6G6D) were first labeledwith the cell staining dye carboxyfluorescein succinimidyl ester (CFSE)and incubated with 75,000 human PBMCs as effector cells at a ratio of5:1 for effector cell: target cell, in the presence of variousconcentrations of Ly6G6D TDB antibodies for 24 hours. At the end of eachassay, live 293 cells were gated out as PI-CFSE+ cells by FACS, andabsolute cell count was obtained by flow cytometry. The percentage ofcell killing was calculated based on non-TDB treated controls. Comparedto the Ly6G6D (40G5c) TDB and Ly6G6D (38E4v1) TDB, the Ly6G6D (38E4v11)TDB displayed enhanced cell killing at lower concentration (FIG. 2).

When tested for their ability to activate the cytotoxic effects of Tcells in in vitro T cell activation assays, the three TDBs againdisplayed varying efficacy. In the T cell activation assays, the293-Ly6G6D cells were incubated for 24 hours with human PBMCs wereisolated from whole blood of healthy donors by Ficoll separation in thepresence of various concentrations of Ly6G6D TDB antibodies, asdescribed above. Activated CD8+ and CD4+ T cells were detected by CD69and CD25 surface expression by flow cytometry analysis. The Ly6G6D(38E4v11) TDB displayed enhanced efficacy in both CD8+ T cell activation(FIG. 3A) and CD4+ T cell activation (FIG. 3B), compared to the othertested Ly6G6D TDBs, particularly the Ly6G6D (40G5c) TDB, which has acomparatively low binding affinity of 51 nM for CD3.

Other Embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An anti-cluster of differentiation 3 (CD3)antibody, wherein the anti-CD3 antibody comprises a binding domaincomprising the following six hypervariable regions (HVRs): (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 1; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO: 2; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 5; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:
 6. 2. The anti-CD3antibody of claim 1, wherein the binding domain comprises (a) a heavychain variable (VH) domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 7;(b) a light chain variable (VL) domain comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 8; or (c) a VH domain as in (a) and a VL domain as in (b).
 3. Theanti-CD3 antibody of claim 2, wherein the VH domain comprises the aminoacid sequence of SEQ ID NO:
 7. 4. The anti-CD3 antibody of claim 2,wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 8.5. An anti-CD3 antibody, wherein the anti-CD3 antibody comprises abinding domain comprising (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 7 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO:
 8. 6. The anti-CD3 antibody of any one of claims1-5, wherein the anti-CD3 antibody binds the human CD3ε polypeptide witha K_(D) of 0.5 nM or lower.
 7. The anti-CD3 antibody of claim 6, whereinthe anti-CD3 antibody binds the human CD3ε polypeptide with a K_(D) of0.3 nM or lower.
 8. The anti-CD3 antibody of claim 7, wherein theanti-CD3 antibody binds the human CD3ε polypeptide with a K_(D) of 0.1nM or lower.
 9. The anti-CD3 antibody of any one of claims 1-5, whereinthe anti-CD3 antibody comprises a substitution mutation in the Fc regionthat reduces effector function.
 10. The anti-CD3 antibody of claim 9,wherein the substitution mutation is an aglycosylation site mutation.11. The anti-CD3 antibody of claim 10, wherein the aglycosylation sitemutation is at amino acid residue N297, L234, L235, and/or D265 (EUnumbering).
 12. The anti-CD3 antibody of claim 11, wherein theaglycosylation site mutation is selected from the group consisting ofN297G, N297A, L234A, L235A, and D265A.
 13. The anti-CD3 antibody of anyone of claims 1-12, wherein the anti-CD3 antibody is monoclonal, human,humanized, or chimeric.
 14. The anti-CD3 antibody of any one of claims1-13, wherein the anti-CD3 antibody is an antibody fragment that bindsCD3.
 15. The anti-CD3 antibody of claim 14, wherein the antibodyfragment is selected from the group consisting of Fab, Fab′-SH, Fv,scFv, and (Fab′)₂ fragments.
 16. The anti-CD3 antibody of any one ofclaims 1-14, wherein the anti-CD3 antibody is a full-length antibody.17. The anti-CD3 antibody of any one of claims 1-16, wherein theanti-CD3 antibody is an IgG antibody.
 18. The anti-CD3 antibody of anyone of claims 1-17, wherein the anti-CD3 antibody is a monospecificantibody.
 19. The anti-CD3 antibody of any one of claims 1-17, whereinthe anti-CD3 antibody is a multispecific antibody.
 20. The anti-CD3antibody of claim 19, wherein the multispecific antibody is a bispecificantibody.
 21. The anti-CD3 antibody of claim 20, wherein the bispecificantibody comprises a second binding domain that binds to a secondbiological molecule, wherein the second biological molecule is a cellsurface antigen on a target cell other than an immune effector cell. 22.The anti-CD3 antibody of claim 21, wherein the cell surface antigen isexpressed in low copy number on the target cell.
 23. The anti-CD3antibody of claim 22, wherein the cell surface antigen is expressed atless than 35,000 copies per target cell.
 24. The anti-CD3 antibody ofclaim 22 or 23, wherein the cell surface antigen is expressed at about100 copies per target cell to about 30,000 copies per target cell. 25.The anti-CD3 antibody of any one of claims 21-24, wherein the cellsurface antigen is a tumor antigen.
 26. The anti-CD3 antibody of claim25, wherein the tumor antigen is Ly6G6D (lymphocyte antigen 6 complex,locus G61).
 27. The anti-CD3 antibody of claim 26, wherein Ly6G6D isexpressed at about 20,000 copies per target cell to about 30,000 copiesper target cell.
 28. An anti-CD3 antibody, wherein the anti-CD3 antibodyis a bispecific antibody that binds to CD3 located on an immune effectorcell and a cell surface antigen that is expressed in low copy number ona target cell other than the immune effector cell, wherein thebispecific antibody comprises an anti-CD3 arm comprising a first bindingdomain comprising the following six HVRs: (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 1; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 2; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 3; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 4; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 5; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 6; and an anti-cell surface antigen armcomprising a second binding domain.
 29. The anti-CD3 antibody of claim28, wherein the cell surface antigen is a tumor antigen.
 30. Theanti-CD3 antibody of claim 29, wherein the tumor antigen is Ly6G6D. 31.The anti-CD3 antibody of any one of claims 1-30, wherein the anti-CD3antibody comprises one or more heavy chain constant domains, wherein theone or more heavy chain constant domains are selected from a first CH1(CH1₁) domain, a first CH2 (CH2₁) domain, a first CH3 (CH3₁) domain, asecond CH1 (CH1₂) domain, second CH2 (CH2₂) domain, and a second CH3(CH3₂) domain.
 32. The anti-CD3 antibody of claim 31, wherein at leastone of the one or more heavy chain constant domains is paired withanother heavy chain constant domain.
 33. The anti-CD3 antibody of claim32, wherein the CH3₁ and CH3₂ domains each comprise a protuberance orcavity, and wherein the protuberance or cavity in the CH3, domain ispositionable in the cavity or protuberance, respectively, in the CH3₂domain.
 34. The anti-CD3 antibody of claim 33, wherein the CH3₁ and CH3₂domains meet at an interface between said protuberance and cavity. 35.The anti-CD3 antibody of any one of claims 31-34, wherein the CH2₁ andCH2₂ domains each comprise a protuberance or cavity, and wherein theprotuberance or cavity in the CH2, domain is positionable in the cavityor protuberance, respectively, in the CH2₂ domain.
 36. The anti-CD3antibody of claim 35, wherein the CH2₁ and CH2₂ domains meet at aninterface between said protuberance and cavity.
 37. An isolated nucleicacid encoding the anti-CD3 antibody of any one of claims 1-36.
 38. Avector comprising the isolated nucleic acid of claim
 37. 39. A host cellcomprising the vector of claim
 38. 40. The host cell of claim 39,wherein the host cell is a mammalian cell.
 41. The host cell of claim40, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.42. The host cell of claim 39, wherein the host cell is a prokaryoticcell.
 43. The host cell of claim 42, wherein the prokaryotic cell is E.coli.
 44. A method of producing the anti-CD3 antibody of any one ofclaims 1-36, the method comprising culturing the host cell of claim 39in a culture medium.
 45. The method of claim 44, wherein the methodfurther comprises recovering the anti-CD3 antibody from the host cell orthe culture medium.
 46. An immunoconjugate comprising the anti-CD3antibody of any one of claims 1-36 and a cytotoxic agent.
 47. Acomposition comprising the anti-CD3 antibody of any one of claims 1-36.48. The composition of claim 47, further comprising a pharmaceuticallyacceptable carrier, excipient, or diluent.
 49. The composition of claim48, wherein the composition is a pharmaceutical composition.
 50. Thecomposition of any one of claims 47-49, wherein the composition furthercomprises a PD-1 axis binding antagonist or an additional therapeuticagent.
 51. The anti-CD3 antibody of any one of claims 1-36 for use as amedicament.
 52. The anti-CD3 antibody of any one of claims 1-36 for usein treating or delaying progression of a cell proliferative disorder oran autoimmune disorder in a subject in need thereof.
 53. The anti-CD3antibody of any one of claims 1-36 for use in enhancing immune functionin a subject having a cell proliferative disorder or an autoimmunedisorder.
 54. The anti-CD3 antibody of claim 52 or 53, wherein the cellproliferative disorder is a cancer.
 55. The anti-CD3 antibody of claim54, wherein the cancer is selected from the group consisting ofesophageal cancer, stomach cancer, small intestine cancer, largeintestine cancer, colorectal cancer, breast cancer, non-small cell lungcancer, non-Hodgkin's lymphoma (NHL), B cell lymphoma, B cell leukemia,multiple myeloma, renal cancer, prostate cancer, liver cancer, head andneck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma,germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC)DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acutemyeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zonelymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacyticlymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervoussystem lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocyticleukemia, Splenic marginal zone lymphoma, Hairy cell leukemia, Spleniclymphoma/leukemia, unclassifiable, Splenic diffuse red pulp small B-celllymphoma, Hairy cell leukemia variant, WaldenstrOm macroglobulinemia,Heavy chain diseases, a Heavy chain disease, γ Heavy chain disease, μHeavy chain disease, Plasma cell myeloma, Solitary plasmacytoma of bone,Extraosseous plasmacytoma, Extranodal marginal zone lymphoma ofmucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zonelymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicularlymphoma, Primary cutaneous follicle centre lymphoma, T-cell/histiocyterich large B-cell lymphoma, Primary DLBCL of the CNS, Primary cutaneousDLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associatedwith chronic inflammation, Lymphomatoid granulomatosis, Primarymediastinal (thymic) large B-cell lymphoma, Intravascular large B-celllymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma,Large B-cell lymphoma arising in HHV8-associated multicentric Castlemandisease, Primary effusion lymphoma: B-cell lymphoma, unclassifiable,with features intermediate between diffuse large B-cell lymphoma andBurkitt lymphoma, and B-cell lymphoma, unclassifiable, with featuresintermediate between diffuse large B-cell lymphoma and classical Hodgkinlymphoma.
 56. The anti-CD3 antibody of claim 55, wherein the cancer isesophageal cancer.
 57. The anti-CD3 antibody of claim 54, wherein thecancer is an adenocarcinoma.
 58. The anti-CD3 antibody of claim 57,wherein the adenocarcinoma is metastatic adenocarcinoma.
 59. Theanti-CD3 antibody of claim 57 or 58, wherein the adenocarcinoma is acolorectal adenocarcinoma, a gastric adenocarcinoma, or a pancreaticadenocarcinoma.
 60. The anti-CD3 antibody of claim 52 or 53, wherein theautoimmune disorder is selected from the group consisting of rheumatoidarthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus(SLE), Wegener's disease, inflammatory bowel disease, idiopathicthrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura(TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgAnephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis,diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome,glomerulonephritis, Neuromyelitis Optica (NMO) and IgG neuropathy. 61.Use of the anti-CD3 antibody of any one of claims 1-36 in themanufacture of a medicament for treating or delaying progression of acell proliferative disorder or an autoimmune disorder.
 62. Use of theanti-CD3 antibody of any one of claims 1-36 in the manufacture of amedicament for enhancing immune function in a subject having a cellproliferative disorder or an autoimmune disorder.
 63. The use of claim61 or 62, wherein the cell proliferative disorder is a cancer.
 64. Theuse of claim 63, wherein the cancer is selected from the groupconsisting of esophageal cancer, stomach cancer, small intestine cancer,large intestine cancer, colorectal cancer, breast cancer, non-small celllung cancer, non-Hodgkin's lymphoma (NHL), B cell lymphoma, B cellleukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer,head and neck cancer, melanoma, ovarian cancer, mesothelioma,glioblastoma, germinal-center B-cell-like (GCB) DLBCL, activatedB-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma(MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL),marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL),lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM),central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cellprolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cellleukemia, Splenic lymphoma/leukemia, unclassifiable, Splenic diffuse redpulp small B-cell lymphoma, Hairy cell leukemia variant, WaldenstrOmmacroglobulinemia, Heavy chain diseases, a Heavy chain disease, γ Heavychain disease, μ Heavy chain disease, Plasma cell myeloma, Solitaryplasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginalzone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma),Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma,Pediatric follicular lymphoma, Primary cutaneous follicle centrelymphoma, T-cell/histiocyte rich large B-cell lymphoma, Primary DLBCL ofthe CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of theelderly, DLBCL associated with chronic inflammation, Lymphomatoidgranulomatosis, Primary mediastinal (thymic) large B-cell lymphoma,Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma,Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, Primary effusion lymphoma: B-celllymphoma, unclassifiable, with features intermediate between diffuselarge B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma,unclassifiable, with features intermediate between diffuse large B-celllymphoma and classical Hodgkin lymphoma.
 65. The use of claim 64,wherein the cancer is esophageal cancer.
 66. The use of claim 63,wherein the cancer is an adenocarcinoma.
 67. The use of claim 66,wherein the adenocarcinoma is metastatic adenocarcinoma.
 68. The use ofclaim 66 or 67, wherein the adenocarcinoma is a colorectaladenocarcinoma, a gastric adenocarcinoma, or a pancreaticadenocarcinoma.
 69. The use of claim 61 or 62, wherein the autoimmunedisorder is selected from the group consisting of rheumatoid arthritis,juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE),Wegener's disease, inflammatory bowel disease, idiopathicthrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura(TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgAnephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis,diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome,glomerulonephritis, Neuromyelitis Optica (NMO) and IgG neuropathy.
 70. Amethod of treating or delaying the progression of a cell proliferativedisorder or an autoimmune disorder in a subject in need thereof, themethod comprising administering to the subject the anti-CD3 antibody ofany one of claims 1-36.
 71. A method of enhancing immune function in asubject having a cell proliferative disorder or an autoimmune disorder,the method comprising administering to the subject an effective amountof the anti-CD3 antibody of any one of claims 1-36.
 72. The method ofclaim 70 or 71, wherein the cell proliferative disorder is a cancer. 73.The method of claim 72, wherein the cancer is selected from the groupconsisting of esophageal cancer, stomach cancer, small intestine cancer,large intestine cancer, colorectal cancer, breast cancer, non-small celllung cancer, non-Hodgkin's lymphoma (NHL), B cell lymphoma, B cellleukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer,head and neck cancer, melanoma, ovarian cancer, mesothelioma,glioblastoma, germinal-center B-cell-like (GCB) DLBCL, activatedB-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma(MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL),marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL),lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM),central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cellprolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cellleukemia, Splenic lymphoma/leukemia, unclassifiable, Splenic diffuse redpulp small B-cell lymphoma, Hairy cell leukemia variant, WaldenstrOmmacroglobulinemia, Heavy chain diseases, a Heavy chain disease, γ Heavychain disease, μ Heavy chain disease, Plasma cell myeloma, Solitaryplasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginalzone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma),Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma,Pediatric follicular lymphoma, Primary cutaneous follicle centrelymphoma, T-cell/histiocyte rich large B-cell lymphoma, Primary DLBCL ofthe CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of theelderly, DLBCL associated with chronic inflammation, Lymphomatoidgranulomatosis, Primary mediastinal (thymic) large B-cell lymphoma,Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma,Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, Primary effusion lymphoma: B-celllymphoma, unclassifiable, with features intermediate between diffuselarge B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma,unclassifiable, with features intermediate between diffuse large B-celllymphoma and classical Hodgkin lymphoma.
 74. The method of claim 73,wherein the cancer is esophageal cancer.
 75. The method of claim 72,wherein the cancer is an adenocarcinoma.
 76. The method of claim 75,wherein the adenocarcinoma is metastatic adenocarcinoma.
 77. The methodof claim 75 or 76, wherein the adenocarcinoma is a colorectaladenocarcinoma, a gastric adenocarcinoma, or a pancreaticadenocarcinoma.
 78. The method of claim 70 or 71, wherein the autoimmunedisorder is selected from the group consisting of rheumatoid arthritis,juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE),Wegener's disease, inflammatory bowel disease, idiopathicthrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura(TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgAnephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis,diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome,glomerulonephritis, Neuromyelitis Optica (NMO) and IgG neuropathy. 79.The method of any one of claims 70-78, wherein the anti-CD3 antibodybinds to (a) a CD3 molecule located on an immune effector cell and (b) asecond biological molecule located on a target cell other than theimmune effector cell.
 80. The method of claim 79, wherein the anti-CD3antibody activates the immune effector cell following binding to (a) and(b).
 81. The method of claim 80, wherein the activated immune effectorcell is capable of exerting a cytotoxic effect and/or an apoptoticeffect on the target cell.
 82. The method of any one of claims 70-81,wherein the anti-CD3 antibody is administered to the subject in a dosageof about 0.01 mg/kg to about 10 mg/kg.
 83. The method of claim 82,wherein the anti-CD3 antibody is administered to the subject in a dosageof about 0.1 mg/kg to about 10 mg/kg.
 84. The method of claim 83,wherein the anti-CD3 antibody is administered to the subject in a dosageof about 1 mg/kg.
 85. The method of any one of claims 70-84, furthercomprising administering to the subject a PD-1 axis binding antagonistand/or an additional therapeutic agent.
 86. The method of claim 85,wherein the PD-1 axis binding antagonist or additional therapeutic agentis administered prior to or subsequent to the administration of theanti-CD3 antibody.
 87. The method of claim 85, wherein the PD-1 axisbinding antagonist additional therapeutic agent is administeredconcurrently with the anti-CD3 antibody.
 88. The method of any one ofclaims 85-87, wherein the PD-1 axis binding antagonist is selected fromthe group consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist.
 89. The method of claim 88,wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.90. The method of claim 89, wherein the PD-1 binding antagonist isselected from the group consisting of MDX 1106 (nivolumab), MK-3475(pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,REGN2810, and BGB-108.
 91. The method of claim 88, wherein the PD-1 axisbinding antagonist is a PD-L1 binding antagonist.
 92. The method ofclaim 91, wherein the PD-L1 binding antagonist is selected from thegroup consisting of MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105,MEDI4736 (durvalumab), and MSB0010718C (avelumab).
 93. The method ofclaim 88, wherein the PD-1 axis binding antagonist is a PD-L2 bindingantagonist.
 94. The method of claim 93, wherein the PD-L2 bindingantagonist is an antibody or an immunoadhesin.
 95. A method of treatingor delaying the progression of a cell proliferative disorder or anautoimmune disorder in a subject in need thereof, the method comprisingadministering to the subject an anti-CD3 antibody and a PD-1 axisbinding antagonist, wherein the anti-CD3 antibody comprises an anti-CD3arm and an anti-Ly6G6D arm.
 96. A method of enhancing immune function ina subject having a cell proliferative disorder or an autoimmunedisorder, the method comprising administering to the subject an anti-CD3antibody and a PD-1 axis binding antagonist, wherein the anti-CD3antibody comprises an anti-CD3 arm and an anti-Ly6G6D arm.
 97. Themethod of claim 95 or 96, wherein: (a) the anti-CD3 arm comprises afirst binding domain comprising: (i) a VH domain comprising an aminoacid sequence of SEQ ID NO: 7, and (ii) a VL domain comprising an aminoacid sequence of SEQ ID NO: 8; and (b) the PD-1 axis binding antagonistis an anti-PD-L1 antibody.
 98. The method of any one of claims 95-97,wherein the cell proliferative disorder is a cancer.
 99. The method ofclaim 98, wherein the cancer is esophageal cancer.
 100. The method ofclaim 98, wherein the cancer is an adenocarcinoma.
 101. The method ofclaim 100, wherein the adenocarcinoma is metastatic adenocarcinoma. 102.The method of claim 100 or 101, wherein the adenocarcinoma is acolorectal adenocarcinoma, a gastric adenocarcinoma, or a pancreaticadenocarcinoma.
 103. The method of any one of claims 70-102, furthercomprising administering to the subject a glucocorticoid.
 104. Themethod of claim 103, wherein the glucocorticoid is dexamethasone. 105.The method of any one of claims 70-104, further comprising administeringto the subject rituximab.
 106. The method of any one of claims 70-104,further comprising administering to the subject obinutuzumab.
 107. Themethod of any one of claims 70-106, further comprising administering tothe subject an antibody-drug conjugate (ADC).
 108. The method of any oneof claims 70-107, wherein the anti-CD3 antibody is administeredsubcutaneously, intravenously, intramuscularly, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally.
 109. Themethod of claim 108, wherein the anti-CD3 antibody is administeredsubcutaneously.
 110. The method of claim 108, wherein the anti-CD3antibody is administered intravenously.
 111. The method of any one ofclaims 70-110, wherein the subject is human.
 112. A kit comprising: (a)the composition of any one of claims 47-50; and (b) a package insertcomprising instructions for administering the composition to a subjectto treat or delay progression of a cell proliferative disorder or anautoimmune disorder.